Methods of using compositions comprising variants and fusions of fgf19 polypeptides for reducing glucose levels in a subject

ABSTRACT

The invention relates to variants and fusions of fibroblast growth factor 19 (FGF19), variants and fusions of fibroblast growth factor 21 (FGF21), fusions of fibroblast growth factor 19 (FGF19) and/or fibroblast growth factor 21 (FGF21), and variants or fusions of fibroblast growth factor 19 (FGF19) and/or fibroblast growth factor 21 (FGF21) proteins and peptide sequences (and peptidomimetics), having one or more activities, such as glucose lowering activity, and methods for and uses in treatment of hyperglycemia and other disorders.

RELATED APPLICATIONS

This application is a division of U.S. Ser. No. 14/743,851, filed Jun.18, 2015, which is a division of U.S. Ser. No. 14/616,401, filed Feb. 6,2015 (now U.S. Pat. No. 9,089,525), which is a division of U.S. Ser. No.13/538,705 filed Jun. 29, 2012 (now U.S. Pat. No. 8,951,966), whichclaims the benefit of priority of U.S. Ser. No. 61/504,128 filed Jul. 1,2011, and U.S. Ser. No. 61/515,126 filed Aug. 4, 2011, each of which areexpressly incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to variants of fibroblast growth factor 19 (FGF19)proteins and peptide sequences (and peptidomimetics) and fusions offibroblast growth factor 19 (FGF19) and/or fibroblast growth factor 21(FGF21) proteins and peptide sequences (and peptidomimetics), andvariants of fusions of fibroblast growth factor 19 (FGF19) and/orfibroblast growth factor 21 (FGF21) proteins and peptide sequences (andpeptidomimetics) having glucose lowering activity, and methods for anduses in treatment of hyperglycemia and other disorders.

INTRODUCTION

Diabetes mellitus is a debilitating metabolic disease caused by absentinsulin production (type 1) or insulin resistance or insufficientinsulin production (type 2) from pancreatic β-cells. β-cells arespecialized endocrine cells that manufacture and store insulin forrelease following a meal. Insulin is a hormone that facilitates thetransfer of glucose from the blood into tissues where it is needed.Patients with diabetes must frequently monitor blood glucose levels andmany require multiple daily insulin injections to survive. However, suchpatients rarely attain ideal glucose levels by insulin injection(Turner, R. C. et al. JAMA 281:2005 (1999)). Furthermore, prolongedelevation of insulin levels can result in detrimental side effects suchas hypoglycemic shock and desensitization of the body's response toinsulin. Consequently, diabetic patients still develop long-termcomplications, such as cardiovascular diseases, kidney disease,blindness, nerve damage and wound healing disorders (UK ProspectiveDiabetes Study (UKPDS) Group, Lancet 352:837 (1998)).

Bariatric surgery has been proposed as a potential treatment fordiabetes. It has been postulated that changes in gut hormone secretionafter the surgery are responsible for the resolution of diabeticconditions. The underlying molecular mechanism has yet to be elucidated,although glucagon-like peptide 1 (GLP-1) has been speculated as apossible candidate (Rubino, F. Diabetes Care 32 Suppl 2:S368 (2009)).FGF19 is highly expressed in the distal small intestine and transgenicover-expression of FGF19 improves glucose homeostasis (Tomlinson, E.Endocrinology 143(5):1741-7 (2002)). Serum levels of FGF19 in humans areelevated following gastric bypass surgery. Augmented expression andsecretion of FGF19 could at least partially explain the diabetesremission experienced following surgery.

Accordingly, there is a need for alternative treatments of hyperglycemicconditions such as diabetes, prediabetes, insulin resistance,hyperinsulinemia, glucose intolerance or metabolic syndrome, and otherdisorders and diseases associated with elevated glucose levels, inhumans. The invention satisfies this need and provides relatedadvantages.

SUMMARY

The invention is based, in part, on variants of fibroblast growth factor19 (FGF19) peptide sequences, fusions of fibroblast growth factor 19(FGF19) and/or fibroblast growth factor 21 (FGF21) peptide sequences andvariants of fusions (chimeras) of fibroblast growth factor 19 (FGF19)and/or fibroblast growth factor 21 (FGF21) peptide sequences having oneor more activities, such as glucose lowering activity. Such variants andfusions (chimeras) of FGF19 and/or FGF21 peptide sequences includesequences that do not increase or induce hepatocellular carcinoma (HCC)formation or HCC tumorigenesis. Such variants and fusions (chimeras) ofFGF19 and/or FGF21 peptide sequences also include sequences that do notinduce a substantial elevation or increase in lipid profile.

In one embodiment, a chimeric peptide sequence includes or consists of:an N-terminal region having at least seven amino acid residues, theN-terminal region having a first amino acid position and a last aminoacid position, where the N-terminal region has a DSSPL (SEQ ID NO:121)or DASPH (SEQ ID NO:122) sequence; and a C-terminal region having aportion of FGF19, where the C-terminal region has a first amino acidposition and a last amino acid position, where the C-terminal regionincludes amino acid residues 16-29 of FGF19 (WGDPIRLRHLYTSG; SEQ IDNO:169), and where the W residue corresponds to the first amino acidposition of the C-terminal region.

In another embodiment, a chimeric peptide sequence includes or consistsof: an N-terminal region having a portion of FGF21, where the N-terminalregion has a first amino acid position and a last amino acid position,where the N-terminal region has a GQV sequence, and where the V residuecorresponds to the last amino acid position of the N-terminal region;and a C-terminal region including a portion of FGF19, the C-terminalregion having a first amino acid position and a last amino acidposition, where the C-terminal region includes amino acid residues 21-29of FGF19 (RLRHLYTSG; SEQ ID NO:185), and where the R residue correspondsto the first position of the C-terminal region.

In a further embodiment, a chimeric peptide sequence includes orconsists of any of: an N-terminal region comprising a portion of SEQ IDNO:100 [FGF21], the N-terminal region having a first amino acid positionand a last amino acid position, wherein the N-terminal region comprisesat least 5 (or more) contiguous amino acids of SEQ ID NO:100 [FGF21]including the amino acid residues GQV, and wherein the V residuecorresponds to the last amino acid position of the N-terminal region;and a C-terminal region comprising a portion of SEQ ID NO:99 [FGF19],the C-terminal region having a first amino acid position and a lastamino acid position, wherein the C-terminal region comprises amino acidresidues 21-29 of SEQ ID NO:99 [FGF19], RLRHLYTSG (SEQ ID NO:185), andwherein the R residue corresponds to the first position of theC-terminal region. In particular aspects, the N-terminal regioncomprises at least 6 contiguous amino acids (or more, e.g., 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-40, 40-50,50-75, 75-100 contiguous amino acids) of SEQ ID NO:100 [FGF21] includingthe amino acid residues GQV.

In an additional embodiment, a peptide sequence includes or consists ofany of: a fibroblast growth factor 19 (FGF19) sequence variant havingone or more amino acid substitutions, insertions or deletions comparedto a reference or wild type FGF19; a fibroblast growth factor 21 (FGF21)sequence variant having one or more amino acid substitutions, insertionsor deletions compared to a reference or wild type FGF21; a portion of anFGF19 sequence fused to a portion of an FGF21 sequence; or a portion ofan FGF19 sequence fused to a portion of an FGF21 sequence, wherein theFGF19 and/or FGF21 sequence portion(s) have one or more amino acidsubstitutions, insertions or deletions compared to a reference or wildtype FGF19 and/or FGF21.

In still further embodiments, a peptide sequence or a chimeric peptidesequence includes or consists of amino-terminal amino acids 1-16 of SEQID NO:100 [FGF21] fused to carboxy-terminal amino acids 21-194 of SEQ IDNO:99 [FGF19], or the peptide sequence has amino-terminal amino acids1-147 of SEQ ID NO:99 [FGF19] fused to carboxy-terminal amino acids147-181 of SEQ ID NO:100 [FGF21] (M41), or the peptide sequence hasamino-terminal amino acids 1-20 of SEQ ID NO:99 [FGF19] fused tocarboxy-terminal amino acids 17-181 of SEQ ID NO:100 [FGF21] (M44), orthe peptide sequence has amino-terminal amino acids 1-146 of SEQ IDNO:100 [FGF21] fused to carboxy-terminal amino acids 148-194 of SEQ IDNO:99 [FGF19] (M45), or the peptide sequence has amino-terminal aminoacids 1-20 of SEQ ID NO:99 [FGF19] fused to internal amino acids 17-146of SEQ ID NO:100 [FGF21] fused to carboxy-terminal amino acids 148-194of SEQ ID NO:99 [FGF19] (M46).

In yet additional embodiments, a peptide sequence or a chimeric peptidesequence has a WGDPI (SEQ ID NO:170) sequence motif corresponding to theWGDPI (SEQ ID NO:170) sequence of amino acids 16-20 of SEQ ID NO:99[FGF19], or has a substituted, mutated or absent WGDPI (SEQ ID NO:170)sequence motif corresponding to FGF19 WGDPI (SEQ ID NO:170) sequence ofamino acids 16-20 of FGF19, or the WGDPI (SEQ ID NO:170) sequence motifhas one or more amino acids substituted, mutated or absent; or isdistinct from an FGF 19 variant sequence having any of GQV, GDI, WGPI(SEQ ID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQID NO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI(SEQ ID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA(SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQID NO:183) or FGDPI (SEQ ID NO:184) substituted for the FGF19 WGDPI (SEQID NO:170) sequence at amino acids 16-20.

In yet further embodiments, a peptide sequence or a chimeric peptidesequence has an N-terminal region that includes or consists of aminoacid residues VHYG (SEQ ID NO:101), where the N-terminal regioncomprises amino acid residues DASPHVHYG (SEQ ID NO:102), or where theN-terminal region comprises amino acid residues DSSPLVHYG (SEQ IDNO:103), or where the N-terminal region comprises amino acid residuesDSSPLLQ (SEQ ID NO:104), or where the N-terminal region comprises aminoacid residues DSSPLLQFGGQV (SEQ ID NO:105). In particular aspects, the Gcorresponds to the last position of the N-terminal region, or the Qresidue is the last amino acid position of the N-terminal region, or theV residue corresponds to the last position of the N-terminal region.

In still additional embodiments, a peptide sequence or a chimericpeptide sequence has an N-terminal region that includes or consists ofRHPIP (SEQ ID NO:106), where R is the first amino acid position of theN-terminal region; or HPIP (SEQ ID NO:107) (e.g., where HPIP (SEQ IDNO:107) are the first 4 amino acid residues of the N-terminal region),where H is the first amino acid position of the N-terminal region; orRPLAF (SEQ ID NO:108), where R is the first amino acid position of theN-terminal region; or PLAF (SEQ ID NO:109), where P is the first aminoacid position of the N-terminal region; or R, where R is the first aminoacid position of the N-terminal region, or has at the N-terminal regionany one of the following sequences: MDSSPL (SEQ ID NO:110), MSDSSPL (SEQID NO:111), SDSSPL (SEQ ID NO:112), MSSPL (SEQ ID NO:113) or SSPL (SEQID NO:114).

In other embodiments, a peptide sequence or a chimeric peptide sequencehas, at the first position of the N-terminal region, an “M” residue, an“R” residue, a “S” residue, a “H” residue, a “P” residue, a “L” residueor an “D” residue. In alternative embodiments, a peptide sequence or achimeric peptide sequence does not have a “M” residue or an “R” residueat the first amino acid position of the N-terminal region.

In still other embodiments, a peptide sequence or a chimeric peptidesequence has at the first and second positions of the N-terminal regionan MR sequence, or at the first and second positions of the N-terminalregion an RM sequence, or at the first and second positions of theN-terminal region an RD sequence, or at the first and second positionsof the N-terminal region an DS sequence, or at the first and secondpositions of the N-terminal region an MD sequence, or at the first andsecond positions of the N-terminal region an MS sequence, or at thefirst through third positions of the N-terminal region an MDS sequence,or at the first through third positions of the N-terminal region an RDSsequence, or at the first through third positions of the N-terminalregion an MSD sequence, or at the first through third positions of theN-terminal region an MSS sequence, or at the first through thirdpositions of the N-terminal region an DSS sequence, or at the firstthrough fourth positions of the N-terminal region an RDSS (SEQ IDNO:115) sequence, or at the first through fourth positions of theN-terminal region an MDSS (SEQ ID NO:116) sequence, or at the firstthrough fifth positions of the N-terminal region an MRDSS (SEQ IDNO:117) sequence, or at the first through fifth positions of theN-terminal region an MSSPL (SEQ ID NO:113) sequence, or at the firstthrough sixth positions of the N-terminal region an MDSSPL (SEQ IDNO:110) sequence, or at the first through seventh positions of theN-terminal region an MSDSSPL (SEQ ID NO:111) sequence.

In still other embodiments, a peptide sequence or a chimeric peptidesequence an addition of amino acid residues 30-194 of SEQ ID NO:99[FGF19] at the C-terminus, resulting in a chimeric polypeptide having atthe last position of the C-terminal region that corresponds to aboutresidue 194 of SEQ ID NO:99 [FGF19]. In further other embodiments, achimeric peptide sequence or peptide sequence comprises all or a portionof an FGF19 sequence (e.g., SEQ ID NO:99), positioned at the C-terminusof the peptide, or where the amino terminal “R” residue is deleted fromthe peptide.

In more particular embodiments, a chimeric peptide sequence or peptidesequence includes or consists of any of M1-M98 variant peptidesequences, or a subsequence or fragment of any of the M1-M98 (SEQ IDNOS:1-52, 192, and 54-98, respectively variant peptide sequences.

In additional particular embodiments, a chimeric peptide sequence orpeptide sequence has an N-terminal or a C-terminal region from about 20to about 200 amino acid residues in length. In further particularembodiments, a chimeric peptide sequence or peptide sequence has atleast one amino acid deletion. In still further particular embodiments,a chimeric peptide sequence or peptide sequence, or a subsequence orfragment thereof, has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or more amino acid deletions from the amino terminus,the carboxy-terminus or internally. In a particular non-limiting aspect,the amino acid substitution, or deletion is at any of amino acidpositions 8-20 of FGF19 (AGPHVHYGWGDPI; SEQ ID NO:187).

In more particular embodiments, a chimeric peptide sequence or peptidesequence includes or consists of an amino acid sequence of about 5 to10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to90, 90 to 100 or more amino acids. In more particular embodiments, achimeric peptide sequence or peptide sequence includes or consists of anamino acid sequence of about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more aminoacids of FGF19 or FGF21.

In further particular embodiments, chimeric peptide sequences andpeptide sequences have particular functions or activities. In oneaspect, a chimeric peptide sequence or peptide sequence maintains orincreases an FGFR4 mediated activity. In additional aspects, a chimericpeptide sequence or peptide sequence binds to fibroblast growth factorreceptor 4 (FGFR4) or activates FGFR4, or does not detectably bind tofibroblast growth factor receptor 4 (FGFR4) or activate FGFR4, or bindsto FGFR4 with an affinity less than, comparable to or greater than FGF19binding affinity for FGFR4, or activates FGFR4 to an extent or amountless than, comparable to or greater than FGF19 activates FGFR4. Infurther aspects, a chimeric peptide sequence or peptide sequence hasreduced hepatocellular carcinoma (HCC) formation compared to FGF19, oran FGF 19 variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171),WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI,WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177),WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180),WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) orFGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ ID NO:170) sequenceat amino acids 16-20 of FGF19, and/or has greater glucose loweringactivity compared to FGF19, or an FGF 19 variant sequence having any ofGQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ IDNO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ IDNO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ IDNO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ IDNO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ ID NO:184) substituted forthe WGDPI (SEQ ID NO:170) sequence at amino acids 16-20 of FGF19, and/orhas less lipid increasing activity compared to FGF19, or an FGF 19variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV(SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI(SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19, and/or has less triglyceride, cholesterol, non-HDLor HDL increasing activity compared to FGF19, or an FGF 19 variantsequence having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ IDNO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ IDNO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ IDNO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ IDNO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19, and/or has less lean mass reducing activitycompared to FGF21. Such functions and activities can be ascertained invitro or in vivo, for example, in a db/db mouse.

In still additional embodiments, chimeric peptide sequences and peptidesequences isolated or purified, and/or chimeric peptide sequences andpeptide sequences can be included in compositions. In one embodiment, achimeric peptide sequence or peptide sequence is included in apharmaceutical composition. Such compositions include combinations ofinactive or other active ingredients. In one embodiment, a compositions,such as a pharmaceutical composition includes chimeric peptide sequenceor peptide sequence and a glucose lowering agent.

In yet further embodiments, nucleic acid molecules encoding the chimericpeptide sequence or peptide sequence are provided. Such molecules canfurther include an expression control element in operable linkage thatconfers expression of the nucleic acid molecule encoding the peptide invitro, in a cell or in vivo, or a vector comprising the nucleic acidmolecule (e.g., a viral vector). Transformed and host cells that expressthe chimeric peptide sequences and peptide sequences are also provided.

Uses and methods of treatment that include administration or delivery ofany chimeric peptide sequence or peptide sequence are also provided. Inparticular embodiments, a use or method of treatment of a subjectincludes administering an invention chimeric peptide or peptide sequenceto a subject, such as a subject having, or at risk of having, a diseaseor disorder treatable by an invention peptide sequence, in an amounteffective for treating the disease or disorder. In a further embodiment,a method includes administering an invention chimeric peptide or peptidesequence to a subject, such as a subject having a hyperglycemiccondition (e.g., diabetes, such as insulin-dependent (type I) diabetes,type II diabetes, or gestational diabetes), insulin resistance,hyperinsulinemia, glucose intolerance or metabolic syndrome, or is obeseor has an undesirable body mass.

In particular aspects of the methods and uses, a chimeric peptidesequence or peptide sequence is administered to a subject in an amounteffective to improve glucose metabolism in the subject. In moreparticular aspects, a subject has a fasting plasma glucose level greaterthan 100 mg/dl or has a hemoglobin A1c (HbA1c) level above 6%, prior toadministration.

In further embodiments, a use or method of treatment of a subject isintended to or results in reduced glucose levels, increased insulinsensitivity, reduced insulin resistance, reduced glucagon, animprovement in glucose tolerance, or glucose metabolism or homeostasis,improved pancreatic function, or reduced triglyceride, cholesterol, IDL,LDL or VLDL levels, or a decrease in blood pressure, a decrease inintimal thickening of the blood vessel, or a decrease in body mass orweight gain.

Methods of analyzing and/or identifying a chimeric peptide sequence orpeptide sequence are also provided, such as chimeric peptide sequencesand peptide sequences that have glucose lowering activity withoutsubstantial hepatocellular carcinoma (HCC) activity. In one embodiment,a method includes: a) providing a candidate chimeric peptide sequence orpeptide sequence; b) administering the candidate peptide sequence to atest animal (e.g., a db/db mouse); c) measuring glucose levels of theanimal after administration of the candidate peptide sequence, todetermine if the candidate peptide sequence reduces glucose levels. In aparticular aspect, the chimeric peptide sequence or peptide sequence isalso analyzed for induction of HCC in the animal (e.g., assessing ahepatic tissue sample from the test animal), or expression of a markercorrelating with HCC activity, wherein a candidate peptide havingglucose lowering activity and not substantial HCC activity. Such methodsidentify the candidate as having glucose lowering activity, optionallyalso without substantial hepatocellular carcinoma (HCC) activity.

DESCRIPTION OF DRAWINGS

FIG. 1 shows representative domain exchanges between FGF21 (no shading)and FGF19 (grey shading) protein sequences, and the resultant fusion(chimeric) sequences. The amino acid regions from each of FGF21 andFGF19 present in the fusion (chimera) are indicated by the numbers.Glucose lowering and lipid elevation are shown for each of the chimericsequences.

FIGS. 2A-2I show glucose lowering and body weight data. A) variant M5;B) variant M1; C) variant M2 and variant M69; D) variant M3; E) variantM48 and variant M49; F) variant M51 and variant M50; G) variant M52peptide; H) variant M53 peptide; and I) variant M70 peptide sequencesall have glucose lowering (i.e., anti-diabetic) activity in db/db mice.Mice were injected with AAV vector expressing FGF19, FGF21, the selectedvariants, and saline and GFP are negative controls.

FIGS. 3A-3I show serum lipid profile (triglyceride, total cholesterol,HDL and non-HDL) of db/db mice injected with AAV vector expressingFGF19, FGF21 or A) variant M5; B) variant M1; C) variant M2 and variantM69; D) variant M3; E) variant M48 and variant M49; F) variant M51 andvariant M50; G) variant M52 peptide; H) variant M53 peptide; and I)variant M70 peptide sequences. Variant M5 peptide sequence did notincrease or elevate lipids, in contrast to FGF19, M1, M2 and M69 whichincreases and elevates lipids. Serum levels of all variants werecomparable. Saline and GFP are negative controls.

FIGS. 4A-4I show hepatocellular carcinoma (HCC)-related data for A)variant M5; B) variant M1; C) variant M2 and variant M69; D) variant M3;E) variant M48 and variant M49; F) variant M51 and variant M50; G)variant M52; H) variant M53 peptide; and I) variant M70 peptidesequences. All variants did not significantly increase or inducehepatocellular carcinoma (HCC) formation or HCC tumorigenesis, incontrast to FGF19. HCC score is recorded as the number of HCC nodules onthe surface of the entire liver from variants-injected mice divided bythe number of HCC nodules from wild type FGF19-injected mice.

FIGS. 5A-5I show lean mass or fat mass data for A) variant M5; B)variant M1; C) variant M2 and variant M69; D) variant M3; E) variant M48and variant M49; F) variant M51 and variant M50; G) variant M52; H)variant M53 peptide; and I) variant M70 peptide sequences. Except forM2, M5 and M69, the variant peptide sequences reduce lean mass or fatmass, in contrast to FGF21.

FIGS. 6A-6B show graphical data demonstrating that injection of therecombinant A) variant M5; and B) variant M69 polypeptides reduce bloodglucose in ob/ob mice.

FIG. 7 shows data indicating that liver expression of aldo-ketoreductase family 1, member C18 (Akr1C18) and solute carrier family 1,member 2 (slc1a2) appears to correlate with HCC activity.

DETAILED DESCRIPTION

The invention provides chimeric and peptide sequences that are able tolower or reduce levels of glucose. In one embodiment, a chimeric peptidesequence includes or consists of an N-terminal region having at leastseven amino acid residues and the N-terminal region having a first aminoacid position and a last amino acid position, where the N-terminalregion has a DSSPL (SEQ ID NO:121) or DASPH (SEQ ID NO:122) sequence;and a C-terminal region having a portion of FGF19 and the C-terminalregion having a first amino acid position and a last amino acidposition, where the C-terminal region includes amino acid residues 16-29of FGF19 (WGDPIRLRHLYTSG; SEQ ID NO:169) and the W residue correspondsto the first amino acid position of the C-terminal region.

In another embodiment, a chimeric peptide sequence includes or consistsof an N-terminal region having a portion of FGF21 and the N-terminalregion having a first amino acid position and a last amino acidposition, where the N-terminal region has a GQV sequence and the Vresidue corresponds to the last amino acid position of the N-terminalregion; and a C-terminal region having a portion of FGF19 and theC-terminal region having a first amino acid position and a last aminoacid position where the C-terminal region includes amino acid residues21-29 of FGF19 (RLRHLYTSG; SEQ ID NO:185) and the R residue correspondsto the first position of the C-terminal region.

In further embodiments, a peptide sequence includes or consists of afibroblast growth factor 19 (FGF19) sequence variant having one or moreamino acid substitutions, insertions or deletions compared to areference or wild type FGF19. In additional embodiments, a peptidesequence includes or consists of a fibroblast growth factor 21 (FGF21)sequence variant having one or more amino acid substitutions, insertionsor deletions compared to a reference or wild type FGF21. In yetadditional embodiments, a peptide sequence includes or consists of aportion of an FGF19 sequence fused to a portion of an FGF21 sequence. Instill additional embodiments, a peptide sequence includes or consists ofa portion of an FGF19 sequence fused to a portion of an FGF21 sequence,where the FGF19 and/or FGF21 sequence portion(s) have one or more aminoacid substitutions, insertions or deletions compared to a reference orwild type FGF19 and/or FGF21.

The invention also provides methods and uses of treating a subjecthaving or at risk of having a metabolic disorder treatable usingvariants and fusions of fibroblast growth factor 19 (FGF19) and/orfibroblast growth factor 21 (FGF21) peptide sequences. In oneembodiment, a method includes contacting or administering to a subjectone or more variant or fusion fibroblast growth factor 19 (FGF19) and/orfibroblast growth factor 21 (FGF21) peptide sequences in an amounteffective for treating the disorder. In another embodiment, a methodincludes contacting or administering to a subject one or more nucleicacid molecules encoding a variant or fusion fibroblast growth factor 19(FGF19) and/or fibroblast growth factor 21 (FGF21) peptide sequence (forexample, an expression control element in operable linkage with thenucleic acid encoding the peptide sequence, optionally including avector), in an amount effective for treating the disorder.

Although an understanding of the underlying mechanism of action of theinvention peptides is not required in order to practice the invention,without being bound to any particular theory or hypothesis, it isbelieved that invention peptides mimic, at least in part, the effectthat bariatric surgery has on, for example, glucose homeostasis andweight loss. Changes in gastrointestinal hormone secretion (e.g.,glucagon-like peptide 1 (GLP-1)) after bariatric surgery are believedresponsible for the resolution of, for example, diabetic conditions.FGF19 is highly expressed in the distal small intestine, and transgenicover-expression of FGF19 improves glucose homeostasis. Because levels ofFGF19 in humans are also elevated following gastric bypass surgery, theelevated FGF19 might be involved with the remission of diabetes observedfollowing bariatric surgery.

A representative reference or wild type FGF19 sequence is set forth as:

(SEQ ID NO: 99) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK.

A representative reference or wild type FGF21 sequence is set forth as:

(SEQ ID NO: 100) HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS.

Representative variant sequences, namely variant M1, variant M2, variantM3, variant M5, variant M48, variant M49, variant M50, variant M51,variant M52, variant M53, variant M69, and variant M70 peptide sequencesare shown below:

(M1; SEQ ID NO: 1)RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK  (M2; SEQ ID NO: 2)RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKEIRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFEK (M3; SEQ ID NO: 3)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK  (M5; SEQ ID NO: 5)RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK (M48; SEQ ID NO: 48)RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK(M49; SEQ ID NO: 49)RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR SPSFEK (M50; SEQ ID NO: 50)RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK (M51; SEQ ID NO: 51)RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK (M52; SEQ ID NO: 52)RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK(M53; SEQ ID NO: 53)MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK(M69; SEQ ID NO: 69)RDSSPLVHYGWGDPIRLRHYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK (M70; SEQ ID NO: 70)MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK 

Three additional allelic (polymorphic) forms of FGF21, namely M71, M72and M73 are also shown below:

(M71; SEQ ID NO: 71) HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS  (M72; SEQ ID NO: 72)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS  (M73; SEQ ID NO: 73)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPENCKTLLTDIDRTH TEKPVWDGITGE FGF21 allelic variants are illustrated above (e.g., M70, M71 and M72).

The terms “peptide,” “protein,” and “polypeptide” sequence are usedinterchangeably herein to refer to two or more amino acids, or“residues,” including chemical modifications and derivatives of aminoacids, covalently linked by an amide bond or equivalent. The amino acidsforming all or a part of a peptide may be from among the known 21naturally occurring amino acids, which are referred to by both theirsingle letter abbreviation or common three-letter abbreviation. In thepeptide sequences of the invention, conventional amino acid residueshave their conventional meaning. Thus, “Leu” is leucine, “Ile” isisoleucine, “Nle” is norleucine, and so on.

Exemplified herein are peptide sequences, distinct from reference FGF19and FGF21 polypeptides set forth herein, that reduce or lower glucose,in vivo (Tables 1-8 and FIG. 1 SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51,52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52,M53, M69, M70, M71, M72 and M73, respectively)). Non-limiting particularexamples are a peptide sequence with amino-terminal amino acids 1-16 ofFGF21 fused to carboxy-terminal amino acids 21-194 of FGF19; a peptidesequence with amino-terminal amino acids 1-147 of FGF19 fused tocarboxy-terminal amino acids 147-181 of FGF21; a peptide sequence withamino-terminal amino acids 1-20 of FGF19 fused to carboxy-terminal aminoacids 17-181 of FGF21; a peptide sequence with amino-terminal aminoacids 1-146 of FGF21 fused to carboxy-terminal amino acids 148-194 ofFGF19; and a peptide sequence with amino-terminal amino acids 1-20 ofFGF19 fused to internal amino acids 17-146 of FGF21 fused tocarboxy-terminal amino acids 148-194 of FGF19.

Additional particular peptides sequences have a WGDPI (SEQ ID NO:170)sequence motif corresponding to the WGDPI (SEQ ID NO:170) sequence ofamino acids 16-20 of FGF19 (SEQ ID NO:99), lack a WGDPI sequence motifcorresponding to the WGDPI (SEQ ID NO:170) sequence of amino acids 16-20of FGF19 (SEQ ID NO:99), or have a substituted (i.e., mutated) WGDPI(SEQ ID NO:170) sequence motif corresponding to FGF19 WGDPI (SEQ IDNO:170) sequence of amino acids 16-20 of FGF19 (SEQ ID NO:99).

Particular peptide sequences of the invention also include sequencesdistinct from FGF19 and FGF21 (e.g., as set forth herein), and FGF 19variant sequences having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV(SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI(SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for FGF19 WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20. Accordingly, the wild-type FGF19 and FGF21 (e.g., as setforth herein as SEQ ID NOS:99 and 100, respectively) may be excludedsequences, and FGF19 having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV(SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI(SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19 may also be excluded. This exclusion, however, doesnot apply to where a sequence has, for example, 3 FGF21 residues fusedto FGF19 having, for example, any of GQV, GQV, GDI, or GPI, or 2 FGF21residues fused to any of WGPI (SEQ ID NO:171), WGDI (SEQ ID NO:173),GDPI (SEQ ID NO:174), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), orWGDP (SEQ ID NO:183).

Particular non-limiting examples of peptide sequences include or consistof all or a part of a sequence variant specified herein as M1-M98 (SEQID NOs:1-52, 192, and 54-98, respectively). More particular non-limitingexamples of peptide sequences include or consist of all or a part of asequence set forth as:

HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK (SEQID NO:160) (FGF21 sequences can also include an “R” residue at the aminoterminus), or a subsequence or fragment thereof; orDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ IDNO:161), or a subsequence or fragment thereof; orRPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE AVRSPSFEK(M1) (SEQ ID NO:1), or a subsequence or fragment thereof; orRPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFEK(M2) (SEQ ID NO:2), or a subsequence or fragment thereof; orDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQID NO:141), or a subsequence or fragment thereof; orRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK(M69) (SEQ ID NO:69), or a subsequence or fragment thereof; orRDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M52)(SEQ ID NO: 52), or a subsequence or fragment thereof; orHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQID NO:160), or a subsequence or fragment thereof;HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M71) (SEQ IDNO:71), or a subsequence or fragment thereof; orHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M72) (SEQ IDNO:72), or a subsequence or fragment thereof; orHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPENCKTLLTDIDRTHTEKPVWDGITGE (M73) (SEQ ID NO:73), or a subsequence orfragment thereof; orRPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE AVRSPSFEK(M1) (SEQ ID NO:1), or a subsequence or fragment thereof; orRPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFEK(M2) (SEQ ID NO:2), or a subsequence or fragment thereof; orRPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE AVRSPSFEK(M3) (SEQ ID NO:3), or a subsequence or fragment thereof; orRDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M48)(SEQ ID NO:48), or a subsequence or fragment thereof; orRPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR SPSFEK(M49) (SEQ ID NO:49), or a subsequence or fragment thereof; orRHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK(M50) (SEQ ID NO:50), or a subsequence or fragment thereof; orRHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK(M51) (SEQ ID NO:51), or a subsequence or fragment thereof; orMDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M53)(SEQ ID NO:192), or a subsequence or fragment thereof; andMRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRS PSFEK(M70) (SEQ ID NO:70), or a subsequence or fragment thereof, or for anyof the foregoing peptide sequences the R terminal residue may bedeleted.

Further particular non-limiting examples of peptide sequences include orconsist of:

HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSP SFEK (SEQID NO:160), or a subsequence or fragment thereof; orDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ IDNO:161), or a subsequence or fragment thereof;RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLE AVRSPSFEK(SEQ ID NO:1), or a subsequence or fragment thereof;RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEA VRSPSFEK(SEQ ID NO:2), or a subsequence or fragment thereof;DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQID NO:141), or a subsequence or fragment thereof.

Additional particular non-limiting examples of peptide sequences, havingat the N-terminus, a peptide sequence including or consisting of all ora part of any of:

(amino acids 1-25 of SEQ ID NO: 160) HPIPDSSPLLQFGGQVRLRHLYTSG (M5-R);(amino acids 2-22 of SEQ ID NO: 6) DSSPLLQFGGQVRLRHLYTSG (M6-R);(amino acids 1-27 of SEQ ID NO: 7) RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7);(amino acids 2-26 of SEQ ID NO: 8) HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R);(amino acids 2-28 of SEQ ID NO: 9) HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R);(amino acids 2-28 of SEQ ID NO: 10)HPIPDSSPHVHYGWGDPI RLRHLYTSG (M10-R);(amino acids 1-27 of SEQ ID NO: 11) RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11);(amino acids 1-29 of SEQ ID NO: 12) RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12);(amino acids 1-27 of SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13);(amino acids 2-26 of SEQ ID NO: 14) HPIPDSSPHVHYGGQVRLRHLYTSG (M14-R);(amino acids 1-27 of SEQ ID NO: 15) RPLAFSDAGPHVHWGDPIRLRHLYTSG (M15);(amino acids 1-27 of SEQ ID NO: 16) RPLAFSDAGPHVHWGDPI RLRHLYTSG (M16);(amino acids 1-27 of SEQ ID NO: 17) RPLAFSDAGPHVGWGDPI RLRHLYTSG (M17);(amino acids 1-27 of SEQ ID NO: 18) RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18);(amino acids 1-27 of SEQ ID NO: 19) RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19);(amino acids 1-27 of SEQ ID NO: 20) RPLAFSDAGPVHGWGDPI RLRHLYTSG (M20);(amino acids 1-27 of SEQ ID NO: 21) RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21);(amino acids 1-27 of SEQ ID NO: 22) RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22);(amino acids 1-27 of SEQ ID NO: 23) RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23);(amino acids 1-27 of SEQ ID NO: 24) RPLAFSDAGPHHYWGDPIRLRHLYTSG (M24);(amino acids 1-27 of SEQ ID NO: 25) RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25);(amino acids 1-27 of SEQ ID NO: 26) RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26);(amino acids 1-27 of SEQ ID NO: 27) RPLAFSDSSPHVHWGDPIRLRHLYTSG (M2722);(amino acids 1-26 of SEQ ID NO: 28) RPLAFSDAGPHVWGDPIRLRHLYTSG (M28);(amino acids 1-28 of SEQ ID NO: 29) RPLAFSDAGPHVHYWGDPI RLRHLYTSG (M29);(amino acids 1-29 of SEQ ID NO: 30) RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30);(amino acids 1-26 of SEQ ID NO: 31) RHPIPDSSPLLQFGAQVRLRHLYTSG (M31);(amino acids 1-26 of SEQ ID NO: 32) RHPIPDSSPLLQFGDQVRLRHLYTSG (M32);(amino acids 1-26 of SEQ ID NO: 33) RHPIPDSSPLLQFGPQVRLRHLYTSG (M33);(amino acids 1-26 of SEQ ID NO: 34) RHPIPDSSPLLQFGGAVRLRHLYTSG (M34);(amino acids 1-26 of SEQ ID NO: 35) RHPIPDSSPLLQFGGEVRLRHLYTSG (M35);(amino acids 1-26 of SEQ ID NO: 36) RHPIPDSSPLLQFGGNVRLRHLYTSG (M36);(amino acids 1-26 of SEQ ID NO: 37) RHPIPDSSPLLQFGGQARLRHLYTSG (M37);(amino acids 1-26 of SEQ ID NO: 38) RHPIPDSSPLLQFGGQI RLRHLYTSG (M38);(amino acids 1-26 of SEQ ID NO: 39) RHPIPDSSPLLQFGGQTRLRHLYTSG (M39);(amino acids 1-28 of SEQ ID NO: 40) RHPIPDSSPLLQFGWGQPVRLRHLYTSG (M40);(amino acids 2-24 of SEQ ID NO: 74) DAGPHVHYGWGDPIRLRHLYTSG (M74-R);(amino acids 2-19 of SEQ ID NO: 75) VHYGWGDPIRLRHLYTSG (M75-R); (amino acids 2-10 of SEQ ID NO: 77) RLRHLYTSG (M77-R); (amino acids 1-28 of SEQ ID NO: 9) RHPIPDSSPLLQFGWGDPIRLRHLYTSG (M9); (amino acids 1-26 of SEQ ID NO: 8) RHPIPDSSPLLQWGDPIRLRHLYTSG (M8); (amino acids 1-29 of SEQ ID NO: 12)RPLAFSDAGPLLQFGWGDPI RLRHLYTSG (M12); (amino acids 1-28 of SEQ ID NO: 10) RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10);(amino acids 1-27 of SEQ ID NO: 13) RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13);(amino acids 1-26 of SEQ ID NO: 14) RHPIPDSSPHVHYGGQVRLRHLYTSG (M14);amino acids 1-27 of SEQ ID NO: 43) RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43);(amino acids 1-22 of SEQ ID NO: 6) RDSSPLLQFGGQVRLRHLYTSG (M6);and for any of the foregoing peptide sequences the amino terminal Rresidue may be deleted.

Peptide sequences of the invention additionally include those withreduced or absent induction or formation of hepatocellular carcinoma(HCC) compared to FGF19, or an FGF 19 variant sequence having any ofGQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ IDNO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ IDNO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ IDNO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ IDNO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ ID NO:184) substituted forthe WGDPI (SEQ ID NO:170) sequence at amino acids 16-20 of FGF19.Peptide sequences of the invention also include those with greaterglucose lowering activity compared to FGF19, or an FGF 19 variantsequence having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ IDNO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ IDNO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ IDNO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ IDNO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19. Peptide sequences of the invention moreoverinclude those with less lipid (e.g., triglyceride, cholesterol, non-HDLor HDL) increasing activity compared to FGF19, or an FGF 19 variantsequence having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ IDNO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ IDNO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ IDNO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ IDNO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19.

Typically, the number of amino acids or residues in an invention peptidesequence will total less than about 250 (e.g., amino acids or mimeticsthereof). In various particular embodiments, the number of residuescomprise from about 20 up to about 200 residues (e.g., amino acids ormimetics thereof). In additional embodiments, the number of residuescomprise from about 50 up to about 200 residues (e.g., amino acids ormimetics thereof). In further embodiments, the number of residuescomprise from about 100 up to about 195 residues (e.g., amino acids ormimetics thereof) in length.

Amino acids or residues can be linked by amide or by non-natural andnon-amide chemical bonds including, for example, those formed withglutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, orN,N′-dicyclohexylcarbodiimide (DCC). Non-amide bonds include, forexample, ketomethylene, aminomethylene, olefin, ether, thioether and thelike (see, e.g., Spatola in Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, Vol. 7, pp 267-357 (1983), “Peptide and BackboneModifications,” Marcel Decker, NY). Thus, when a peptide of theinvention includes a portion of an FGF19 sequence and a portion of anFG21 sequence, the two portions need not be joined to each other by anamide bond, but can be joined by any other chemical moiety or conjugatedtogether via a linker moiety.

The invention also includes subsequences, variants and modified forms ofthe exemplified peptide sequences (including the FGF19 and FGF21variants and subsequences listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3,5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48,M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73, respectively), andthe FGF19/FGF21 fusions and chimeras listed in Tables 1-8 and SEQ IDNOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2,M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73,respectively)), so long as the foregoing retains at least a detectableor measureable activity or function. For example, certain exemplifiedvariant peptides have FGF19 C-terminal sequence,PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLS SAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO:188) at theC-terminal portion, e.g., following the “TSG” amino acid residues of thevariant.

Also, certain exemplified variant peptides, for example, those havingall or a portion of FGF21 sequence at the amino-terminus, have an “R”residue positioned at the N-terminus, which can be omitted. Similarly,certain exemplified variant peptides, include an “M” residue positionedat the N-terminus, which can be appended to or further substituted foran omitted residue, such as an “R” residue. More particularly, invarious embodiments peptide sequences at the N-terminus include any of:RDSS (SEQ ID NO:115), DSS, MDSS (SEQ ID NO:116) or MRDSS (SEQ IDNO:117). Furthermore, in cells when a “M” residue is adjacent to a “S”residue, the “M” residue may be cleaved such that the “M” residue isdeleted from the peptide sequence, whereas when the “M” residue isadjacent to a “D” residue, the “M” residue may not be cleaved. Thus, byway of example, in various embodiments peptide sequences include thosewith the following residues at the N-terminus: MDSSPL (SEQ ID NO:119),MSDSSPL (SEQ ID NO:120) (cleaved to SDSSPL (SEQ ID NO:112)) and MSSPL(SEQ ID NO:113) (cleaved to SSPL (SEQ ID NO:114)).

Accordingly, the “peptide,” “polypeptide,” and “protein” sequences ofthe invention include subsequences, variants and modified forms of theFGF19 and FGF21 variants and subsequences listed in Tables 1-8 and SEQID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1,M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73,respectively), and the FGF19/FGF21 fusions and chimeras listed in Tables1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72and M73, respectively), so long as the subsequence, variant or modifiedform (e.g., fusion or chimera) retains at least a detectable activity orfunction.

As used herein, the term “modify” and grammatical variations thereof,means that the composition deviates relative to a reference composition,such as a peptide sequence. Such modified peptide sequences, nucleicacids and other compositions may have greater or less activity orfunction, or have a distinct function or activity compared with areference unmodified peptide sequence, nucleic acid, or othercomposition, or may have a property desirable in a protein formulatedfor therapy (e.g. serum half-life), to elicit antibody for use in adetection assay, and/or for protein purification. For example, a peptidesequence of the invention can be modified to increase serum half-life,to increase in vitro and/or in vivo stability of the protein, etc.

Particular examples of such subsequences, variants and modified forms ofthe peptide sequences exemplified herein (e.g., a peptide sequencelisted in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53,69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53,M69, M70, M71, M72 and M73, respectively)) include substitutions,deletions and/or insertions/additions of one or more amino acids, to orfrom the amino terminus, the carboxy-terminus or internally. One exampleis a substitution of an amino acid residue for another amino acidresidue within the peptide sequence. Another is a deletion of one ormore amino acid residues from the peptide sequence, or an insertion oraddition of one or more amino acid residues into the peptide sequence.

The number of residues substituted, deleted or inserted/added are one ormore amino acids (e.g., 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140,140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, 225-250,or more) of a peptide sequence. Thus, an FGF19 or FGF21 sequence canhave few or many amino acids substituted, deleted or inserted/added(e.g., 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80,80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160,160-170, 170-180, 180-190, 190-200, 200-225, 225-250, or more). Inaddition, an FGF19 amino acid sequence can include or consist of anamino acid sequence of about 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140,140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, 225-250,or more amino acids from FGF21; or an FGF21 amino acid or sequence caninclude or consist of an amino acid sequence of about 1-3, 3-5, 5-10,10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110,110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190,190-200, 200-225, 225-250, or more amino acids from FGF19.

Specific examples of substitutions include substituting a D residue foran L-residue. Accordingly, although residues are listed in the L-isomerconfiguration D-amino acids at any particular or all positions of thepeptide sequences of the invention are included, unless a D-isomer leadsto a sequence that has no detectable or measurable function.

Additional specific examples are non-conservative and conservativesubstitutions. A “conservative substitution” is a replacement of oneamino acid by a biologically, chemically or structurally similarresidue. Biologically similar means that the substitution is compatiblewith a biological activity, e.g., glucose lowering activity.Structurally similar means that the amino acids have side chains withsimilar length, such as alanine, glycine and serine, or having similarsize, or the structure of a first, second or additional peptide sequenceis maintained. Chemical similarity means that the residues have the samecharge or are both hydrophilic and hydrophobic. Particular examplesinclude the substitution of one hydrophobic residue, such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, serinefor threonine, etc. Routine assays can be used to determine whether asubsequence, variant or modified form has activity, e.g., glucoselowering activity.

Particular examples of subsequences, variants and modified forms of thepeptide sequences exemplified herein (e.g., a peptide sequence listed inTables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70,71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70,M71, M72 and M73, respectively)) have 50%-60%, 60%-70%, 70%-75%,75%-80%, 80%-85%, 85%-90%, 90%-95%, or 96%, 97%, 98%, or 99% identity toa reference peptide sequence (for example, a peptide sequence in any ofTables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70,71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70,M71, M72 and M73, respectively)). The term “identity” and “homology” andgrammatical variations thereof mean that two or more referenced entitiesare the same. Thus, where two amino acid sequences are identical, theyhave the identical amino acid sequence. “Areas, regions or domains ofidentity” mean that a portion of two or more referenced entities are thesame. Thus, where two amino acid sequences are identical or homologousover one or more sequence regions, they share identity in these regions.

The extent of identity between two sequences can be ascertained using acomputer program and mathematical algorithm known in the art. Suchalgorithms that calculate percent sequence identity (homology) generallyaccount for sequence gaps and mismatches over the comparison region. Forexample, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschulet al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI)has exemplary search parameters as follows: Mismatch −2; gap open 5; gapextension 2. For peptide sequence comparisons, a BLASTP algorithm istypically used in combination with a scoring matrix, such as PAM100, PAM250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCHsequence comparison programs are also used to quantitate the extent ofidentity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988);Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol.Biol. 147:195 (1981)). Programs for quantitating protein structuralsimilarity using Delaunay-based topological mapping have also beendeveloped (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

In the invention peptide sequences, including subsequences, variants andmodified forms of the peptide sequences exemplified herein (e.g.,sequences listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50,51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51,M52, M53, M69, M70, M71, M72 and M73, respectively)) an “amino acid” or“residue” includes conventional alpha-amino acids as well as beta-aminoacids, alpha, alpha disubstituted amino acids and N-substituted aminoacids wherein at least one side chain is an amino acid side chain moietyas defined herein. An “amino acid” further includes N-alkyl alpha-aminoacids, wherein the N-terminus amino group has a C₁ to C₆ linear orbranched alkyl substituent. The term “amino acid” therefore includesstereoisomers and modifications of naturally occurring protein aminoacids, non-protein amino acids, post-translationally modified aminoacids (e.g., by glycosylation, phosphorylation, ester or amide cleavage,etc.), enzymatically modified or synthesized amino acids, derivatizedamino acids, constructs or structures designed to mimic amino acids,amino acids with a side chain moiety modified, derivatized fromnaturally occurring moieties, or synthetic, or not naturally occurring,etc. Modified and unusual amino acids are included in the peptidesequences of the invention (see, for example, in Synthetic Peptides: AUser's Guide; Hruby et al., Biochem. J. 268:249 (1990); and Toniolo C.,Int. J. Peptide Protein Res. 35:287 (1990)).

In addition, protecting and modifying groups of amino acids areincluded. The term “amino acid side chain moiety” as used hereinincludes any side chain of any amino acid, as the term “amino acid” isdefined herein. This therefore includes the side chain moiety innaturally occurring amino acids. It further includes side chain moietiesin modified naturally occurring amino acids as set forth herein andknown to one of skill in the art, such as side chain moieties instereoisomers and modifications of naturally occurring protein aminoacids, non-protein amino acids, post-translationally modified aminoacids, enzymatically modified or synthesized amino acids, derivatizedamino acids, constructs or structures designed to mimic amino acids,etc. For example, the side chain moiety of any amino acid disclosedherein or known to one of skill in the art is included within thedefinition.

A “derivative of an amino acid side chain moiety” is included within thedefinition of an amino acid side chain moiety. Non-limiting examples ofderivatized amino acid side chain moieties include, for example: (a)adding one or more saturated or unsaturated carbon atoms to an existingalkyl, aryl, or aralkyl chain; (b) substituting a carbon in the sidechain with another atom, preferably oxygen or nitrogen; (c) adding aterminal group to a carbon atom of the side chain, including methyl(—CH₃), methoxy (—OCH₃), nitro (—NO₂), hydroxyl (—OH), or cyano (—C═N);(d) for side chain moieties including a hydroxy, thiol or amino groups,adding a suitable hydroxy, thiol or amino protecting group; or (e) forside chain moieties including a ring structure, adding one or more ringsubstituents, including hydroxyl, halogen, alkyl, or aryl groupsattached directly or through an ether linkage. For amino groups,suitable protecting groups are known to the skilled artisan. Providedsuch derivatization provides a desired activity in the final peptidesequence (e.g., glucose lowering, improved glucose or lipid metabolism,anti-diabetic activity, absence of substantial HCC formation ortumorigenesis, absence of substantial modulation of lean or fat mass,etc.).

An “amino acid side chain moiety” includes all such derivatization, andparticular non-limiting examples include: gamma-amino butyric acid,12-amino dodecanoic acid, alpha-aminoisobutyric acid, 6-amino hexanoicacid, 4-(aminomethyl)-cyclohexane carboxylic acid, 8-amino octanoicacid, biphenylalanine, Boc—t-butoxycarbonyl, benzyl, benzoyl,citrulline, diaminobutyric acid, pyrrollysine, diaminopropionic acid,3,3-diphenylalanine, orthonine, citrulline,1,3-dihydro-2H-isoindolecarboxylic acid, ethyl,Fmoc—fluorenylmethoxycarbonyl, heptanoyl (CH3-(CH2).sub.5-C(═O)—),hexanoyl (CH3-(CH2)4-C(═O)—), homoarginine, homocysteine, homolysine,homophenylalanine, homoserine, methyl, methionine sulfoxide, methioninesulfone, norvaline (NVA), phenylglycine, propyl, isopropyl, sarcosine(SAR), tert-butylalanine, and benzyloxycarbonyl.

A single amino acid, including stereoisomers and modifications ofnaturally occurring protein amino acids, non-protein amino acids,post-translationally modified amino acids, enzymatically synthesizedamino acids, non-naturally occurring amino acids including derivatizedamino acids, an alpha, alpha disubstituted amino acid derived from anyof the foregoing (i.e., an alpha, alpha disubstituted amino acid,wherein at least one side chain is the same as that of the residue fromwhich it is derived), a beta-amino acid derived from any of theforegoing (i.e., a beta-amino acid which other than for the presence ofa beta-carbon is otherwise the same as the residue from which it isderived) etc., including all of the foregoing can be referred to hereinas a “residue.” Suitable substituents, in addition to the side chainmoiety of the alpha-amino acid, include C1 to C6 linear or branchedalkyl. Aib is an example of an alpha, alpha disubstituted amino acid.While alpha, alpha disubstituted amino acids can be referred to usingconventional L- and D-isomeric references, it is to be understood thatsuch references are for convenience, and that where the substituents atthe alpha-position are different, such amino acid can interchangeably bereferred to as an alpha, alpha disubstituted amino acid derived from theL- or D-isomer, as appropriate, of a residue with the designated aminoacid side chain moiety. Thus (S)-2-Amino-2-methyl-hexanoic acid can bereferred to as either an alpha, alpha disubstituted amino acid derivedfrom L-Nle (norleucine) or as an alpha, alpha disubstituted amino acidderived from D-Ala. Similarly, Aib can be referred to as an alpha, alphadisubstituted amino acid derived from Ala. Whenever an alpha, alphadisubstituted amino acid is provided, it is to be understood asincluding all (R) and (S) configurations thereof.

An “N-substituted amino acid” includes any amino acid wherein an aminoacid side chain moiety is covalently bonded to the backbone amino group,optionally where there are no substituents other than H in thealpha-carbon position. Sarcosine is an example of an N-substituted aminoacid. By way of example, sarcosine can be referred to as anN-substituted amino acid derivative of Ala, in that the amino acid sidechain moiety of sarcosine and Ala is the same, i.e., methyl.

Covalent modifications of the invention peptide sequences, includingsubsequences, variants and modified forms of the peptide sequencesexemplified herein (e.g., sequences listed in Tables 1-8 and SEQ ID NOs:1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3,M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73,respectively)), are included in the invention. One type of covalentmodification includes reacting targeted amino acid residues with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues of the peptide.Derivatization with bifunctional agents is useful, for instance, forcross linking peptide to a water-insoluble support matrix or surface foruse in the method for purifying anti-peptide antibodies, and vice-versa.Commonly used cross linking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains (T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminalamine, amidation of any C-terminal carboxyl group, etc.

Exemplified peptide sequences, and subsequences, variants and modifiedforms of the peptide sequences exemplified herein (e.g., sequenceslisted in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53,69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53,M69, M70, M71, M72 and M73, respectively)), can also include alterationsof the backbone for stability, derivatives, and peptidomimetics. Theterm “peptidomimetic” includes a molecule that is a mimic of a residue(referred to as a “mimetic”), including but not limited to piperazinecore molecules, keto-piperazine core molecules and diazepine coremolecules. Unless otherwise specified, an amino acid mimetic of aninvention peptide sequence includes both a carboxyl group and aminogroup, and a group corresponding to an amino acid side chain, or in thecase of a mimetic of Glycine, no side chain other than hydrogen.

By way of example, these would include compounds that mimic the sterics,surface charge distribution, polarity, etc. of a naturally occurringamino acid, but need not be an amino acid, which would impart stabilityin the biological system. For example, Proline may be substituted byother lactams or lactones of suitable size and substitution; Leucine maybe substituted by an alkyl ketone, N-substituted amide, as well asvariations in amino acid side chain length using alkyl, alkenyl or othersubstituents, others may be apparent to the skilled artisan. Theessential element of making such substitutions is to provide a moleculeof roughly the same size and charge and configuration as the residueused to design the molecule. Refinement of these modifications will bemade by analyzing the compounds in a functional (e.g., glucose lowering)or other assay, and comparing the structure activity relationship. Suchmethods are within the scope of the skilled artisan working in medicinalchemistry and drug development.

Another type of modification of the invention peptide sequences,including subsequences, sequence variants and modified forms of theexemplified peptide sequences (including the peptides listed in Tables1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72and M73, respectively)), is glycosylation. As used herein,“glycosylation” broadly refers to the presence, addition or attachmentof one or more sugar (e.g., carbohydrate) moieties to proteins, lipidsor other organic molecules. The use of the term “deglycosylation” hereinis generally intended to mean the removal or deletion, of one or moresugar (e.g., carbohydrate) moieties. In addition, the phrase includesqualitative changes in the glycosylation of the native proteinsinvolving a change in the type and proportions (amount) of the varioussugar (e.g., carbohydrate) moieties present.

Glycosylation can be achieved by modification of an amino acid residue,or by adding one or more glycosylation sites that may or may not bepresent in the native sequence. For example, a typicallynon-glycosylated residue can be substituted for a residue that may beglycosylated. Addition of glycosylation sites can be accomplished byaltering the amino acid sequence. The alteration to the peptide sequencemay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues (for O-linked glycosylation sites) orasparagine residues (for N-linked glycosylation sites). The structuresof N-linked and O-linked oligosaccharides and the sugar residues foundin each type may be different. One type of sugar that is commonly foundon both is N-acetylneuraminic acid (hereafter referred to as sialicacid). Sialic acid is usually the terminal residue of both N-linked andO-linked oligosaccharides and, by virtue of its negative charge, mayconfer acidic properties to the glycoprotein.

Peptide sequences of the invention may optionally be altered throughchanges at the nucleotide (e.g., DNA) level, particularly by mutatingthe DNA encoding the peptide at preselected bases such that codons aregenerated that will translate into the desired amino acids. Anothermeans of increasing the number of carbohydrate moieties on the peptideis by chemical or enzymatic coupling of glycosides to the polypeptide(see, for example, in WO 87/05330). De-glycosylation can be accomplishedby removing the underlying glycosylation site, by deleting theglycosylation by chemical and/or enzymatic means, or by substitution ofcodons encoding amino acid residues that are glycosylated. Chemicaldeglycosylation techniques are known, and enzymatic cleavage ofcarbohydrate moieties on polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases.

Various cell lines can be used to produce proteins that areglycosylated. One non-limiting example is Dihydrofolate reductase(DHFR)-deficient Chinese Hamster Ovary (CHO) cells, which are a commonlyused host cell for the production of recombinant glycoproteins. Thesecells do not express the enzyme beta-galactosidealpha-2,6-sialyltransferase and therefore do not add sialic acid in thealpha-2,6 linkage to N-linked oligosaccharides of glycoproteins producedin these cells.

Another type of modification is to conjugate (e.g., link) one or moreadditional components or molecules at the N- and/or C-terminus of aninvention peptide sequence, such as another protein (e.g., a proteinhaving an amino acid sequence heterologous to the subject protein), or acarrier molecule. Thus, an exemplary peptide sequence can be a conjugatewith another component or molecule.

In certain embodiments, the amino- or carboxy-terminus of an inventionpeptide sequence can be fused with an immunoglobulin Fc region (e.g.,human Fc) to form a fusion conjugate (or fusion molecule). Fc fusionconjugates can increase the systemic half-life of biopharmaceuticals,and thus the biopharmaceutical product may have prolonged activity orrequire less frequent administration. Fc binds to the neonatal Fcreceptor (FcRn) in endothelial cells that line the blood vessels, and,upon binding, the Fc fusion molecule is protected from degradation andre-released into the circulation, keeping the molecule in circulationlonger. This Fc binding is believed to be the mechanism by whichendogenous IgG retains its long plasma half-life. Well-known andvalidated Fc-fusion drugs consist of two copies of a biopharmaceuticallinked to the Fc region of an antibody to improve pharmacokinetics,solubility, and production efficiency. More recent Fc-fusion technologylinks a single copy of a biopharmaceutical to Fc region of an antibodyto optimize the pharmacokinetic and pharmacodynamic properties of thebiopharmaceutical as compared to traditional Fc-fusion conjugates.

A conjugate modification can be used to produce a peptide sequence thatretains activity with an additional or complementary function oractivity of the second molecule. For example, a peptide sequence may beconjugated to a molecule, e.g., to facilitate solubility, storage, invivo or shelf half-life or stability, reduction in immunogenicity,delayed or controlled release in vivo, etc. Other functions oractivities include a conjugate that reduces toxicity relative to anunconjugated peptide sequence, a conjugate that targets a type of cellor organ more efficiently than an unconjugated peptide sequence, or adrug to further counter the causes or effects associated with a disorderor disease as set forth herein (e.g., diabetes).

Clinical effectiveness of protein therapeutics may be limited by shortplasma half-life and susceptibility to degradation. Studies of varioustherapeutic proteins have shown that various modifications, includingconjugating or linking the peptide sequence to any of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes (see, for example, typicallyvia a linking moiety covalently bound to both the protein and thenonproteinaceous polymer (e.g., a PEG) can prolong half-life. SuchPEG-conjugated biomolecules have been shown to possess clinically usefulproperties, including better physical and thermal stability, protectionagainst susceptibility to enzymatic degradation, increased solubility,longer in vivo circulating half-life and decreased clearance, reducedimmunogenicity and antigenicity, and reduced toxicity.

PEGS suitable for conjugation to an invention peptide sequence isgenerally soluble in water at room temperature, and have the generalformula R(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or a protective groupsuch as an alkyl or an alkanol group, and where n is an integer from 1to 1000. When R is a protective group, it generally has from 1 to 8carbons. The PEG conjugated to the peptide sequence can be linear orbranched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGS areincluded in the invention. A molecular weight of the PEG used in theinvention is not restricted to any particular range, but certainembodiments have a molecular weight between 500 and 20,000 while otherembodiments have a molecular weight between 4,000 and 10,000.

The invention includes compositions of conjugates wherein the PEGS havedifferent “n” values and thus the various different PEGs are present inspecific ratios. For example, some compositions comprise a mixture ofconjugates where n=1, 2, 3 and 4. In some compositions, the percentageof conjugates where n=1 is 18-25%, the percentage of conjugates wheren=2 is 50-66%, the percentage of conjugates where n=3 is 12-16%, and thepercentage of conjugates where n=4 is up to 5%. Such compositions can beproduced by reaction conditions and purification methods know in theart.

PEG may directly or indirectly (e.g., through an intermediate) bind tothe peptide sequences of the invention. For example, in one embodiment,PEG binds via a terminal reactive group (a “spacer”). The spacer, is,for example, a terminal reactive group which mediates a bond between thefree amino or carboxyl groups of one or more of the peptide sequencesand polyethylene glycol. The PEG having the spacer which may be bound tothe free amino group includes N-hydroxysuccinylimide polyethylene glycolwhich may be prepared by activating succinic acid ester of polyethyleneglycol with N-hydroxysuccinylimide. Another activated polyethyleneglycol which may be bound to free amino group is2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine which may beprepared by reacting polyethylene glycol monomethyl ether with cyanuricchloride. The activated polyethylene glycol which is bound to the freecarboxyl group includes polyoxyethylenediamine.

Conjugation of one or more of invention peptide sequences to PEG havinga spacer may be carried out by various conventional methods. Forexample, the conjugation reaction can be carried out in solution at a pHof from 5 to 10, at temperature from 4° C. to room temperature, for 30minutes to 20 hours, utilizing a molar ratio of reagent to protein offrom 4:1 to 30:1. Reaction conditions may be selected to direct thereaction towards producing predominantly a desired degree ofsubstitution. In general, low temperature, low pH (e.g., pH=5), andshort reaction time tend to decrease the number of PEGs attached,whereas high temperature, neutral to high pH (e.g., pH≧7), and longerreaction time tend to increase the number of PEGS attached. Variousmethods known in the art may be used to terminate the reaction. In someembodiments the reaction is terminated by acidifying the reactionmixture and freezing at, e.g., −20° C.

Invention peptide sequences including subsequences, sequence variantsand modified forms of the exemplified peptide sequences (including thepeptides listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50,51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51,M52, M53, M69, M70, M71, M72 and M73, respectively)), further includeconjugation to large, slowly metabolized macromolecules such asproteins; polysaccharides, such as sepharose, agarose, cellulose,cellulose beads; polymeric amino acids such as polyglutamic acid,polylysine; amino acid copolymers; inactivated virus particles;inactivated bacterial toxins such as toxoid from diphtheria, tetanus,cholera, leukotoxin molecules; inactivated bacteria; and dendriticcells. Such conjugated forms, if desired, can be used to produceantibodies against peptide sequences of the invention.

Additional suitable components and molecules for conjugation include,for example, thyroglobulin; albumins such as human serum albumin (HSA);tetanus toxoid; Diphtheria toxoid; polyamino acids such aspoly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses;influenza virus hemaglutinin, influenza virus nucleoprotein; KeyholeLimpet Hemocyanin (KLH); and hepatitis B virus core protein and surfaceantigen; or any combination of the foregoing.

Fusion of albumin to an invention peptide sequence can, for example, beachieved by genetic manipulation, such that the DNA coding for HSA(human serum albumin), or a fragment thereof, is joined to the DNAcoding for a peptide sequence. Thereafter, a suitable host can betransformed or transfected with the fused nucleotide sequence in theform of, for example, a suitable plasmid, so as to express a fusionpolypeptide. The expression may be effected in vitro from, for example,prokaryotic or eukaryotic cells, or in vivo from, for example, atransgenic organism. In some embodiments of the invention, theexpression of the fusion protein is performed in mammalian cell lines,for example, CHO cell lines.

Further means for genetically fusing target proteins or peptides toalbumin include a technology known as Albufuse® (Novozymes BiopharmaA/S; Denmark), and the conjugated therapeutic peptide sequencesfrequently become much more effective with better uptake in the body.The technology has been utilized commercially to produce Albuferon®(Human Genome Sciences), a combination of albumin and interferon α-2Bused to treat hepatitis C infection.

Another embodiment entails the use of one or more human domainantibodies (dAb). dAbs are the smallest functional binding units ofhuman antibodies (IgGs) and have favorable stability and solubilitycharacteristics. The technology entails a dAb(s) conjugated to HSA(thereby forming a “AlbudAb”; see, e.g., EP1517921B, WO2005/118642 andWO2006/051288) and a molecule of interest (e.g., a peptide sequence ofthe invention). AlbudAbs are often smaller and easier to manufacture inmicrobial expression systems, such as bacteria or yeast, than currenttechnologies used for extending the serum half-life of peptides. As HSAhas a half-life of about three weeks, the resulting conjugated moleculeimproves the half-life. Use of the dAb technology may also enhance theefficacy of the molecule of interest.

Additional suitable components and molecules for conjugation includethose suitable for isolation or purification. Particular non-limitingexamples include binding molecules, such as biotin (biotin-avidinspecific binding pair), an antibody, a receptor, a ligand, a lectin, ormolecules that comprise a solid support, including, for example, plasticor polystyrene beads, plates or beads, magnetic beads, test strips, andmembranes.

Purification methods such as cation exchange chromatography may be usedto separate conjugates by charge difference, which effectively separatesconjugates into their various molecular weights. For example, the cationexchange column can be loaded and then washed with ˜20 mM sodiumacetate, pH˜4, and then eluted with a linear (0M to 0.5M) NaCl gradientbuffered at a pH from 3 to 5.5, preferably at pH˜4.5. The content of thefractions obtained by cation exchange chromatography may be identifiedby molecular weight using conventional methods, for example, massspectroscopy, SDS-PAGE, or other known methods for separating molecularentities by molecular weight. A fraction is then accordingly identifiedwhich contains the conjugate having the desired number of PEGS attached,purified free from unmodified protein sequences and from conjugateshaving other numbers of PEGs attached.

In still other embodiments, an invention peptide sequence is linked to achemical agent (e.g., an immunotoxin or chemotherapeutic agent),including, but are not limited to, a cytotoxic agent, including taxol,cytochalasin B, gramicidin D, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, andanalogs or homologs thereof. Other chemical agents include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine); alkylating agents (e.g.,mechlorethamine, carmustine and lomustine, cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cisplatin);antibiotics (e.g., bleomycin); and anti-mitotic agents (e.g.,vincristine and vinblastine). Cytotoxins can be conjugated to a peptideof the invention using linker technology known in the art and describedherein.

Further suitable components and molecules for conjugation include thosesuitable for detection in an assay. Particular non-limiting examplesinclude detectable labels, such as a radioisotope (e.g., ¹²⁵I; ³⁵S, ³²P;³³P), an enzyme which generates a detectable product (e.g., luciferase,β-galactosidase, horse radish peroxidase and alkaline phosphatase), afluorescent protein, a chromogenic protein, dye (e.g., fluoresceinisothiocyanate); fluorescence emitting metals (e.g., ¹⁵²Eu);chemiluminescent compounds (e.g., luminol and acridinium salts);bioluminescent compounds (e.g., luciferin); and fluorescent proteins.Indirect labels include labeled or detectable antibodies that bind to apeptide sequence, where the antibody may be detected.

In certain embodiments, a peptide sequence of the invention isconjugated to a radioactive isotope to generate a cytotoxicradiopharmaceutical (radioimmunoconjugates) useful as a diagnostic ortherapeutic agent. Examples of such radioactive isotopes include, butare not limited to, iodine¹³ indium¹¹¹, yttrium⁹⁰ and lutetium¹⁷⁷.Methods for preparing radioimmunoconjugates are known to the skilledartisan. Examples of radioimmunoconjugates that are commerciallyavailable include ibritumomab, tiuxetan, and tositumomab.

Other means and methods included in the invention for prolonging thecirculation half-life, increasing stability, reducing clearance, oraltering immunogenicity or allergenicity of a peptide sequence of theinvention involves modification of the peptide sequence by hesylation,which utilizes hydroxyethyl starch derivatives linked to other moleculesin order to modify the molecule's characteristics. Various aspects ofhesylation are described in, for example, U.S. Patent Appln. Nos.2007/0134197 and 2006/0258607.

Any of the foregoing components and molecules used to modify peptidesequences of the invention may optionally be conjugated via a linker.Suitable linkers include “flexible linkers” which are generally ofsufficient length to permit some movement between the modified peptidesequences and the linked components and molecules. The linker moleculesare generally about 6-50 atoms long. The linker molecules may also be,for example, aryl acetylene, ethylene glycol oligomers containing 2-10monomer units, diamines, diacids, amino acids, or combinations thereof.Suitable linkers can be readily selected and can be of any suitablelength, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50 aminoacids (e.g., Gly).

Exemplary flexible linkers include glycine polymers (G)n, glycine-serinepolymers (for example, (GS)n, GSGGSn (SEQ ID NO:129) and GGGSn (SEQ IDNO:130), where n is an integer of at least one), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers. Glycineand glycine-serine polymers are relatively unstructured, and thereforemay serve as a neutral tether between components. Exemplary flexiblelinkers include, but are not limited to GGSG (SEQ ID NO:131), GGSGG (SEQID NO:132), GSGSG (SEQ ID NO:133), GSGGG (SEQ ID NO:134), GGGSG (SEQ IDNO:189), and GSSSG (SEQ ID NO:135).

Peptide sequences of the invention, including the FGF19 and FGF21variants and subsequences and the FGF19/FGF21 fusions and chimeraslisted in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53,69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53,M69, M70, M71, M72 and M73, respectively), as well as subsequences,sequence variants and modified forms of the sequences listed in Tables1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72and M73, respectively) have one or more activities as set forth herein.One example of an activity is glucose lowering activity. Another exampleof an activity is reduced stimulation or formation of hepatocellularcarcinoma (HCC), for example, as compared to FGF19. An additionalexample of an activity is lower or reduced lipid (e.g., triglyceride,cholesterol, non-HDL) or HDL increasing activity, for example, ascompared to FGF21. A further example of an activity is a lower orreduced lean muscle mass reducing activity, for example, as compared toFGF21. Yet another example of an activity is binding to fibroblastgrowth factor receptor-4 (FGFR4), or activating FGFR4, for example,peptide sequences that bind to FGFR4 with an affinity comparable to orgreater than FGF19 binding affinity for FGFR4; and peptide sequencesthat activate FGFR4 to an extent or amount comparable to or greater thanFGF19 activates FGFR4. Still further examples of activities includedown-regulation or reduction of aldo-keto reductase gene expression, forexample, compared to FGF19; up-regulation or increased Slc1a2 geneexpression compared to FGF21.

More particularly, peptide sequences of the invention, including theFGF19 and FGF21 variants and subsequences and the FGF19/FGF21 fusionsand chimeras listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49,50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50,M51, M52, M53, M69, M70, M71, M72 and M73, respectively), as well assubsequences, variants and modified forms of the sequences listed inTables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70,71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70,M71, M72 and M73, respectively) include those with the followingactivities: peptide sequences having reduced hepatocellular carcinoma(HCC) formation compared to FGF19, or an FGF 19 variant sequence havingany of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ IDNO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ IDNO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ ID NO:184) substituted forthe WGDPI (SEQ ID NO:170) sequence at amino acids 16-20 of FGF19;peptide sequences having greater glucose lowering activity compared toFGF19, or FGF 19 variant sequence having any of GQV, GDI, WGPI (SEQ IDNO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ IDNO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ IDNO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ IDNO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ IDNO:170) sequence at amino acids 16-20 of FGF19; peptide sequences havingless lipid increasing activity (e.g., less triglyceride, cholesterol,non-HDL) or more HDL increasing activity compared to FGF19, or an FGF 19variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV(SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI(SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19; and peptide sequences having less lean massreducing activity as compared to FGF21.

More particularly, peptide sequences of the invention, including theFGF19 and FGF21 variants and subsequences and the FGF19/FGF21 fusionsand chimeras listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49,50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50,M51, M52, M53, M69, M70, M71, M72 and M73, respectively), as well assubsequences, variants and modified forms of the sequences listed inTables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70,71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70,M71, M72 and M73, respectively) include those with the followingactivities: peptide sequences that bind to fibroblast growth factorreceptor-4 (FGFR4), or activate FGFR4, such as peptide sequences thatbind to FGFR4 with an affinity comparable to or greater than FGF19binding affinity for FGFR4; peptide sequences that activate FGFR4 to anextent or amount comparable to or greater than FGF19 activates FGFR4;peptide sequences that down-regulate or reduce aldo-keto reductase geneexpression, for example, compared to FGF19; and peptide sequences thatup-regulate or increase solute carrier family 1, member 2 (Slc1a2) geneexpression as compared to FGF21.

Activities such as, for example, hepatocellular carcinoma (HCC)formation or tumorigenesis, glucose lowering activity, lipid increasingactivity, or lean mass reducing activity can be ascertained in ananimal, such as a db/db mouse. Measurement of binding to FGFR4 oractivation of FGFR4 can be ascertained by assays disclosed herein (see,for example, Example 1) or known to the skilled artisan.

The term “bind,” or “binding,” when used in reference to a peptidesequence, means that the peptide sequence interacts at the molecularlevel. Thus, a peptide sequence that binds to FGFR4 binds to all or apart of the FGFR4 sequence. Specific and selective binding can bedistinguished from non-specific binding using assays known in the art(e.g., competition binding, immunoprecipitation, ELISA, flow cytometry,Western blotting).

Peptides and peptidomimetics can be produced and isolated using methodsknown in the art. Peptides can be synthesized, in whole or in part,using chemical methods (see, e.g., Caruthers (1980). Nucleic Acids Res.Symp. Ser. 215; Horn (1980); and Banga, A. K., Therapeutic Peptides andProteins, Formulation, Processing and Delivery Systems (1995) TechnomicPublishing Co., Lancaster, Pa.). Peptide synthesis can be performedusing various solid-phase techniques (see, e.g., Roberge Science 269:202(1995); Merrifield, Methods Enzymol. 289:3 (1997)) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the manufacturer's instructions.Peptides and peptide mimetics can also be synthesized usingcombinatorial methodologies. Synthetic residues and polypeptidesincorporating mimetics can be synthesized using a variety of proceduresand methodologies known in the art (see, e.g., Organic SynthesesCollective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY).Modified peptides can be produced by chemical modification methods (see,for example, Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, FreeRadic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886(1994)). Peptide sequence variations, derivatives, substitutions andmodifications can also be made using methods such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR based mutagenesis. Site-directed mutagenesis (Carter et al.,Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res.10:6487 (1987)), cassette mutagenesis (Wells et al., Gene 34:315(1985)), restriction selection mutagenesis (Wells et al., Philos. Trans.R. Soc. London SerA 317:415 (1986)) and other techniques can beperformed on cloned DNA to produce invention peptide sequences,variants, fusions and chimeras, and variations, derivatives,substitutions and modifications thereof.

A “synthesized” or “manufactured” peptide sequence is a peptide made byany method involving manipulation by the hand of man. Such methodsinclude but are not limited to the aforementioned, such as chemicalsynthesis, recombinant DNA technology, biochemical or enzymaticfragmentation of larger molecules, and combinations of the foregoing.

Peptide sequences of the invention including subsequences, sequencevariants and modified forms of the exemplified peptide sequences (e.g.,sequences listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50,51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51,M52, M53, M69, M70, M71, M72 and M73, respectively)), can also bemodified to form a chimeric molecule. In accordance with the invention,there are provided peptide sequences that include a heterologous domain.Such domains can be added to the amino-terminus or at thecarboxyl-terminus of the peptide sequence. Heterologous domains can alsobe positioned within the peptide sequence, and/or alternatively flankedby FGF19 and/or FGF21 derived amino acid sequences.

The term “peptide” also includes dimers or multimers (oligomers) ofpeptides. In accordance with the invention, there are also provideddimers or multimers (oligomers) of the exemplified peptide sequences aswell as subsequences, variants and modified forms of the exemplifiedpeptide sequences (e.g., sequences listed in Tables 1-8 and SEQ ID NOs:1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3,M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73,respectively)).

The invention further provides nucleic acid molecules encoding peptidesequences of the invention, including subsequences, sequence variantsand modified forms of the sequences listed in Tables 1-8 and SEQ ID NOs:1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3,M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73,respectively), and vectors that include nucleic acid that encodes thepeptide. Accordingly, “nucleic acids” include those that encode theexemplified peptide sequences disclosed herein, as well as thoseencoding functional subsequences, sequence variants and modified formsof the exemplified peptide sequences, so long as the foregoing retain atleast detectable or measureable activity or function. For example, asubsequence, a variant or modified form of an exemplified peptidesequence disclosed herein (e.g., a sequence listed in Tables 1-8 and SEQID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1,M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73,respectively)) that retains some ability to lower or reduce glucose,provide normal glucose homeostasis, or reduce the histopathologicalconditions associated with chronic or acute hyperglycemia in vivo, etc.

Nucleic acid, which can also be referred to herein as a gene,polynucleotide, nucleotide sequence, primer, oligonucleotide or proberefers to natural or modified purine- and pyrimidine-containing polymersof any length, either polyribonucleotides or polydeoxyribonucleotides ormixed polyribo-polydeoxyribo nucleotides and α-anomeric forms thereof.The two or more purine- and pyrimidine-containing polymers are typicallylinked by a phosphoester bond or analog thereof. The terms can be usedinterchangeably to refer to all forms of nucleic acid, includingdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleicacids can be single strand, double, or triplex, linear or circular.Nucleic acids include genomic DNA and cDNA. RNA nucleic acid can bespliced or unspliced mRNA, rRNA, tRNA or antisense. Nucleic acidsinclude naturally occurring, synthetic, as well as nucleotide analoguesand derivatives.

As a result of the degeneracy of the genetic code, nucleic acidmolecules include sequences degenerate with respect to nucleic acidmolecules encoding the peptide sequences of the invention. Thus,degenerate nucleic acid sequences encoding peptide sequences, includingsubsequences, variants and modified forms of the peptide sequencesexemplified herein (e.g., sequences listed in Tables 1-8 and SEQ ID NOs:1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3,M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73,respectively)), are provided. The term “complementary,” when used inreference to a nucleic acid sequence, means the referenced regions are100% complementary, i.e., exhibit 100% base pairing with no mismatches.

Nucleic acid can be produced using any of a variety of known standardcloning and chemical synthesis methods, and can be altered intentionallyby site-directed mutagenesis or other recombinant techniques known toone skilled in the art. Purity of polynucleotides can be determinedthrough sequencing, gel electrophoresis, UV spectrometry.

Nucleic acids may be inserted into a nucleic acid construct in whichexpression of the nucleic acid is influenced or regulated by an“expression control element,” referred to herein as an “expressioncassette.” The term “expression control element” refers to one or morenucleic acid sequence elements that regulate or influence expression ofa nucleic acid sequence to which it is operatively linked. An expressioncontrol element can include, as appropriate, promoters, enhancers,transcription terminators, gene silencers, a start codon (e.g., ATG) infront of a protein-encoding gene, etc.

An expression control element operatively linked to a nucleic acidsequence controls transcription and, as appropriate, translation of thenucleic acid sequence. The term “operatively linked” refers to ajuxtaposition wherein the referenced components are in a relationshippermitting them to function in their intended manner. Typically,expression control elements are juxtaposed at the 5′ or the 3′ ends ofthe genes but can also be intronic.

Expression control elements include elements that activate transcriptionconstitutively, that are inducible (i.e., require an external signal orstimuli for activation), or derepressible (i.e., require a signal toturn transcription off; when the signal is no longer present,transcription is activated or “derepressed”). Also included in theexpression cassettes of the invention are control elements sufficient torender gene expression controllable for specific cell-types or tissues(i.e., tissue-specific control elements). Typically, such elements arelocated upstream or downstream (i.e., 5′ and 3′) of the coding sequence.Promoters are generally positioned 5′ of the coding sequence. Promoters,produced by recombinant DNA or synthetic techniques, can be used toprovide for transcription of the polynucleotides of the invention. A“promoter” typically means a minimal sequence element sufficient todirect transcription.

Nucleic acids may be inserted into a plasmid for transformation into ahost cell and for subsequent expression and/or genetic manipulation. Aplasmid is a nucleic acid that can be stably propagated in a host cell;plasmids may optionally contain expression control elements in order todrive expression of the nucleic acid. For purposes of this invention, avector is synonymous with a plasmid. Plasmids and vectors generallycontain at least an origin of replication for propagation in a cell anda promoter. Plasmids and vectors may also include an expression controlelement for expression in a host cell, and are therefore useful forexpression and/or genetic manipulation of nucleic acids encoding peptidesequences, expressing peptide sequences in host cells and organisms(e.g., a subject in need of treatment), or producing peptide sequences,for example.

As used herein, the term “transgene” means a polynucleotide that hasbeen introduced into a cell or organism by artifice. For example, a cellhaving a transgene, the transgene has been introduced by geneticmanipulation or “transformation” of the cell. A cell or progeny thereofinto which the transgene has been introduced is referred to as a“transformed cell” or “transformant.” Typically, the transgene isincluded in progeny of the transformant or becomes a part of theorganism that develops from the cell. Transgenes may be inserted intothe chromosomal DNA or maintained as a self-replicating plasmid, YAC,minichromosome, or the like.

Bacterial system promoters include T7 and inducible promoters such as pLof bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) andtetracycline responsive promoters. Insect cell system promoters includeconstitutive or inducible promoters (e.g., ecdysone). Mammalian cellconstitutive promoters include SV40, RSV, bovine papilloma virus (BPV)and other virus promoters, or inducible promoters derived from thegenome of mammalian cells (e.g., metallothionein IIA promoter; heatshock promoter) or from mammalian viruses (e.g., the adenovirus latepromoter; the inducible mouse mammary tumor virus long terminal repeat).Alternatively, a retroviral genome can be genetically modified forintroducing and directing expression of a peptide sequence inappropriate host cells.

As methods and uses of the invention include in vivo delivery,expression systems further include vectors designed for in vivo use.Particular non-limiting examples include adenoviral vectors (U.S. Pat.Nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S. Pat. No.5,604,090), herpes simplex virus vectors (U.S. Pat. No. 5,501,979),retroviral vectors (U.S. Pat. Nos. 5,624,820, 5,693,508 and 5,674,703),BPV vectors (U.S. Pat. No. 5,719,054), CMV vectors (U.S. Pat. No.5,561,063) and parvovirus, rotavirus, Norwalk virus and lentiviralvectors (see, e.g., U.S. Pat. No. 6,013,516). Vectors include those thatdeliver genes to cells of the intestinal tract, including the stem cells(Croyle et al., Gene Ther. 5:645 (1998); S. J. Henning, Adv. Drug Deliv.Rev. 17:341 (1997), U.S. Pat. Nos. 5,821,235 and 6,110,456). Many ofthese vectors have been approved for human studies.

Yeast vectors include constitutive and inducible promoters (see, e.g.,Ausubel et al., In: Current Protocols in Molecular Biology, Vol. 2, Ch.13, ed., Greene Publish. Assoc. & Wiley Interscience, 1988; Grant et al.Methods in Enzymology, 153:516 (1987), eds. Wu & Grossman; BitterMethods in Enzymology, 152:673 (1987), eds. Berger & Kimmel, Acad.Press, N.Y.; and, Strathern et al., The Molecular Biology of the YeastSaccharomyces (1982) eds. Cold Spring Harbor Press, Vols. I and II). Aconstitutive yeast promoter such as ADH or LEU2 or an inducible promotersuch as GAL may be used (R. Rothstein In: DNA Cloning, A PracticalApproach, Vol. 11, Ch. 3, ed. D. M. Glover, IRL Press, Wash., D.C.,1986). Vectors that facilitate integration of foreign nucleic acidsequences into a yeast chromosome, via homologous recombination forexample, are known in the art. Yeast artificial chromosomes (YAC) aretypically used when the inserted polynucleotides are too large for moreconventional vectors (e.g., greater than about 12 Kb).

Expression vectors also can contain a selectable marker conferringresistance to a selective pressure or identifiable marker (e.g.,beta-galactosidase), thereby allowing cells having the vector to beselected for, grown and expanded. Alternatively, a selectable marker canbe on a second vector that is co-transfected into a host cell with afirst vector containing a nucleic acid encoding a peptide sequence.Selection systems include but are not limited to herpes simplex virusthymidine kinase gene (Wigler et al., Cell 11:223 (1977)),hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al.,Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes thatcan be employed in tk−, hgprt− or aprt− cells, respectively.Additionally, antimetabolite resistance can be used as the basis ofselection for dhfr, which confers resistance to methotrexate (O'Hare etal., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); the gpt gene, whichconfers resistance to mycophenolic acid (Mulligan et al., Proc. Natl.Acad. Sci. USA 78:2072 (1981)); neomycin gene, which confers resistanceto aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1(1981)); puromycin; and hygromycin gene, which confers resistance tohygromycin (Santerre et al., Gene 30:147 (1984)). Additional selectablegenes include trpB, which allows cells to utilize indole in place oftryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman et al., Proc. Natl. Acad. Sci. USA 85:8047 (1988));and ODC (ornithine decarboxylase), which confers resistance to theornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO(McConlogue (1987) In: Current Communications in Molecular Biology, ColdSpring Harbor Laboratory).

In accordance with the invention, there are provided transformed cell(s)(in vitro, ex vivo and in vivo) and host cells that produce a variant orfusion of FGF19 and/or FGF21 as set forth herein, where expression ofthe variant or fusion of FGF19 and/or FGF21 is conferred by a nucleicacid encoding the variant or fusion of FGF19 and/or FGF21. Transformedand host cells that express invention peptide sequences typicallyinclude a nucleic acid that encodes the invention peptide sequence. Inone embodiment, a transformed or host cell is a prokaryotic cell. Inanother embodiment, a transformed or host cell is a eukaryotic cell. Invarious aspects, the eukaryotic cell is a yeast or mammalian (e.g.,human, primate, etc.) cell.

As used herein, a “transformed” or “host” cell is a cell into which anucleic acid is introduced that can be propagated and/or transcribed forexpression of an encoded peptide sequence. The term also includes anyprogeny or subclones of the host cell.

Transformed and host cells include but are not limited to microorganismssuch as bacteria and yeast; and plant, insect and mammalian cells. Forexample, bacteria transformed with recombinant bacteriophage nucleicacid, plasmid nucleic acid or cosmid nucleic acid expression vectors;yeast transformed with recombinant yeast expression vectors; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid); insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus); and animal cell systems infected withrecombinant virus expression vectors (e.g., retroviruses, adenovirus,vaccinia virus), or transformed animal cell systems engineered fortransient or stable propagation or expression.

For gene therapy uses and methods, a transformed cell can be in asubject. A cell in a subject can be transformed with a nucleic acid thatencodes an invention peptide sequence as set forth herein in vivo.Alternatively, a cell can be transformed in vitro with a transgene orpolynucleotide, and then transplanted into a tissue of subject in orderto effect treatment. Alternatively, a primary cell isolate or anestablished cell line can be transformed with a transgene orpolynucleotide that encodes a variant of FGF19 and/or FGF21 or afusion/chimeric sequence (or variant) thereof, such as a chimericpeptide sequence including all or a portion of FGF19, or including allor a portion of FGF21, and then optionally transplanted into a tissue ofa subject.

Non-limiting target cells for expression of peptide sequences,particularly for expression in vivo, include pancreas cells (isletcells), muscle cells, mucosal cells and endocrine cells. Such endocrinecells can provide inducible production (secretion) of a variant of FGF19and/or FGF21, or a fusion/chimeric sequence (or variant) thereof, suchas a chimeric peptide sequence including all or a portion of FGF19, orincluding all or a portion of FGF21. Additional cells to transforminclude stem cells or other multipotent or pluripotent cells, forexample, progenitor cells that differentiate into the various pancreascells (islet cells), muscle cells, mucosal cells and endocrine cells.Targeting stem cells provides longer term expression of peptidesequences of the invention.

As used herein, the term “cultured,” when used in reference to a cell,means that the cell is grown in vitro. A particular example of such acell is a cell isolated from a subject, and grown or adapted for growthin tissue culture. Another example is a cell genetically manipulated invitro, and transplanted back into the same or a different subject.

The term “isolated,” when used in reference to a cell, means a cell thatis separated from its naturally occurring in vivo environment.“Cultured” and “isolated” cells may be manipulated by the hand of man,such as genetically transformed. These terms include any progeny of thecells, including progeny cells that may not be identical to the parentalcell due to mutations that occur during cell division. The terms do notinclude an entire human being.

Nucleic acids encoding invention peptide sequences can be introduced forstable expression into cells of a whole organism. Such organismsincluding non-human transgenic animals are useful for studying theeffect of peptide expression in a whole animal and therapeutic benefit.For example, as disclosed herein, production of a variant of FGF19and/or FGF21 or a fusion/chimeric sequence (or variant) thereof, such asa chimeric peptide sequence including all or a portion of FGF19, orincluding all or a portion of FGF21 as set forth herein, in mice loweredglucose and is anti-diabetic.

Mice strains that develop or are susceptible to developing a particulardisease (e.g., diabetes, degenerative disorders, cancer, etc.) are alsouseful for introducing therapeutic proteins as described herein in orderto study the effect of therapeutic protein expression in the diseasesusceptible mouse. Transgenic and genetic animal models that aresusceptible to particular disease or physiological conditions, such asstreptozotocin (STZ)-induced diabetic (STZ) mice, are appropriatetargets for expressing variants of FGF19 and/or FGF21, fusions/chimericsequences (or variant) thereof, such as a chimeric peptide sequenceincluding all or a portion of FGF19, or including all or a portion ofFGF21, as set forth herein. Thus, in accordance with the invention,there are provided non-human transgenic animals that produce a variantof FGF19 and/or FGF21, or a fusion/chimeric sequence (or variant)thereof, such as a chimeric peptide sequence including all or a portionof FGF19, or including all or a portion of FGF21, the production ofwhich is not naturally occurring in the animal which is conferred by atransgene present in somatic or germ cells of the animal.

The term “transgenic animal” refers to an animal whose somatic or germline cells bear genetic information received, directly or indirectly, bydeliberate genetic manipulation at the subcellular level, such as bymicroinjection or infection with recombinant virus. The term“transgenic” further includes cells or tissues (i.e., “transgenic cell,”“transgenic tissue”) obtained from a transgenic animal geneticallymanipulated as described herein. In the present context, a “transgenicanimal” does not encompass animals produced by classical crossbreedingor in vitro fertilization, but rather denotes animals in which one ormore cells receive a nucleic acid molecule. Invention transgenic animalscan be either heterozygous or homozygous with respect to the transgene.Methods for producing transgenic animals, including mice, sheep, pigsand frogs, are well known in the art (see, e.g., U.S. Pat. Nos.5,721,367, 5,695,977, 5,650,298, and 5,614,396) and, as such, areadditionally included.

Peptide sequences, nucleic acids encoding peptide sequences, vectors andtransformed host cells expressing peptide sequences include isolated andpurified forms. The term “isolated,” when used as a modifier of aninvention composition, means that the composition is separated,substantially completely or at least in part, from one or morecomponents in an environment. Generally, compositions that exist innature, when isolated, are substantially free of one or more materialswith which they normally associate within nature, for example, one ormore protein, nucleic acid, lipid, carbohydrate or cell membrane. Theterm “isolated” does not exclude alternative physical forms of thecomposition, such as variants, modifications or derivatized forms,fusions and chimeras, multimers/oligomers, etc., or forms expressed inhost cells. The term “isolated” also does not exclude forms (e.g.,pharmaceutical compositions, combination compositions, etc.) in whichthere are combinations therein, any one of which is produced by the handof man.

An “isolated” composition can also be “purified” when free of some, asubstantial number of, or most or all of one or more other materials,such as a contaminant or an undesired substance or material. Peptidesequences of the invention are generally not known or believed to existin nature. However, for a composition that does exist in nature, anisolated composition will generally be free of some, a substantialnumber of, or most or all other materials with which it typicallyassociates with in nature. Thus, an isolated peptide sequence that alsooccurs in nature does not include polypeptides or polynucleotidespresent among millions of other sequences, such as proteins of a proteinlibrary or nucleic acids in a genomic or cDNA library, for example. A“purified” composition includes combinations with one or more otherinactive or active molecules. For example, a peptide sequence of theinvention combined with another drug or agent, such as a glucoselowering drug or therapeutic agent, for example.

As used herein, the term “recombinant,” when used as a modifier ofpeptide sequences, nucleic acids encoding peptide sequences, etc., meansthat the compositions have been manipulated (i.e., engineered) in afashion that generally does not occur in nature (e.g., in vitro). Aparticular example of a recombinant peptide would be where a peptidesequence of the invention is expressed by a cell transfected with anucleic acid encoding the peptide sequence. A particular example of arecombinant nucleic acid would be where a nucleic acid (e.g., genomic orcDNA) encoding a peptide sequence cloned into a plasmid, with or without5′, 3′ or intron regions that the gene is normally contiguous with inthe genome of the organism. Another example of a recombinant peptide ornucleic acid is a hybrid or fusion sequence, such as a chimeric peptidesequence comprising a portion of FGF19 and a portion of FGF21.

In accordance with the invention, there are provided compositions andmixtures of invention peptide sequences, including subsequences,variants and modified forms of the exemplified peptide sequences(including the FGF19 and FGF21 variants and subsequences listed inTables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70,71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70,M71, M72 and M73, respectively), and the FGF19/FGF21 fusions andchimeras listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50,51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51,M52, M53, M69, M70, M71, M72 and M73, respectively)). In one embodiment,a mixture includes one or more peptide sequences and a pharmaceuticallyacceptable carrier or excipient. In another embodiment, a mixtureincludes one or more peptide sequences and an adjunct drug ortherapeutic agent, such as an anti-diabetic, or glucose lowering, drugor therapeutic agent. Examples of drugs and therapeutic agents are setforth hereafter. Combinations, such as one or more peptide sequences ina pharmaceutically acceptable carrier or excipient, with one or more ofan anti-diabetic, or glucose lowering drug or therapeutic agent are alsoprovided. Such combinations of peptide sequence of the invention withanother drug or agent, such as a glucose lowering drug or therapeuticagent, for example are useful in accordance with the invention methodsand uses, for example, for treatment of a subject.

Combinations also include incorporation of peptide sequences or nucleicacids of the invention into particles or a polymeric substances, such aspolyesters, carbohydrates, polyamine acids, hydrogel, polyvinylpyrrolidone, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers; entrapment in microcapsules prepared by coacervationtechniques or by interfacial polymerization, for example, by the use ofhydroxymethylcellulose or gelatin-microcapsules, or poly(methylmethacrolate) microcapsules, respectively; incorporation incolloid drug delivery and dispersion systems such as macromoleculecomplexes, nano-capsules, microspheres, beads, and lipid-based systems(e.g., N-fatty acyl groups such as N-lauroyl, N-oleoyl, fatty aminessuch as dodecyl amine, oleoyl amine, etc., see U.S. Pat. No. 6,638,513),including oil-in-water emulsions, micelles, mixed micelles, andliposomes, for example.

Invention peptides including subsequences, variants and modified formsof the exemplified peptide sequences (including the FGF19 and FGF21variants and subsequences listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3,5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48,M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73, respectively), andthe FGF19/FGF21 fusions and chimeras listed in Tables 1-8 and SEQ IDNOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2,M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73,respectively)) as set forth herein can be used to modulate glucosemetabolism and facilitate transport of glucose from the blood to keymetabolic organs such as muscle, liver and fat. Such peptide sequencescan be produced in amounts sufficient or effective to restore glucosetolerance and/or to improve or provide normal glucose homeostasis.

As disclosed herein, administration of various FGF19 and/FGF21 variantsand fusion peptide sequences to mice successfully reduced glucoselevels. Furthermore, in contrast to FGF19, certain peptide sequences didnot stimulate or induce HCC formation or tumorigenesis in mice. Thus,administration of invention peptides, including subsequences, variantsand modified forms of the exemplified peptide sequences (including theFGF19 and FGF21 variants and subsequences listed in Tables 1-8 and SEQID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1,M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73,respectively), and the FGF19/FGF21 fusions and chimeras listed in Tables1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72and M73, respectively)), into an animal, either by direct or indirect invivo or by ex vivo methods (e.g., administering the variant or fusionpeptide, a nucleic acid encoding the variant or fusion peptide, or atransformed cell or gene therapy vector expressing the variant or fusionpeptide), can be used to treat various disorders.

Accordingly, the invention includes in vitro, ex vivo and in vivo (e.g.,on or in a subject) methods and uses. Such methods and uses can bepracticed with any of the peptide sequences of the invention set forthherein.

In accordance with the invention, there are provided methods of treatinga subject having, or at risk of having, a disorder. In variousembodiments, a method includes administering a peptide sequence, such asan FGF19 or FGF21 variant, fusion or chimera listed in Tables 1-8 andSEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73(M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 andM73, respectively), or a subsequence, a variant or modified form of anFGF19 or FGF21 variant, fusion or chimera listed in Tables 1-8 and SEQID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1,M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73,respectively), to a subject in an amount effective for treating thedisorder.

Exemplary disorders treatable, preventable, and the like with inventionpeptides, and methods and uses, include metabolic diseases anddisorders. Non limiting examples of diseases and disorders include: 1.Glucose utilization disorders and the sequelae associated therewith,including diabetes mellitus (Type I and Type-2), gestational diabetes,hyperglycemia, insulin resistance, abnormal glucose metabolism,“pre-diabetes” (Impaired Fasting Glucose (IFG) or Impaired GlucoseTolerance (IGT)), and other physiological disorders associated with, orthat result from, the hyperglycemic condition, including, for example,histopathological changes such as pancreatic β-cell destruction. Fortreatment, invention peptide sequences can be administered to subjectshaving a fasting plasma glucose (FPG) level greater than about 100mg/dl. Peptide sequences of the invention may also be useful in otherhyperglycemic-related disorders, including kidney damage (e.g., tubuledamage or nephropathy), liver degeneration, eye damage (e.g., diabeticretinopathy or cataracts), and diabetic foot disorders; 2. Dyslipidemiasand their sequelae such as, for example, atherosclerosis, coronaryartery disease, cerebrovascular disorders and the like; 3. Otherconditions which may be associated with the metabolic syndrome, such asobesity and elevated body mass (including the co-morbid conditionsthereof such as, but not limited to, nonalcoholic fatty liver disease(NAFLD), nonalcoholic steatohepatitis (NASH), and polycystic ovariansyndrome (PCOS)), and also include thromboses, hypercoagulable andprothrombotic states (arterial and venous), hypertension, cardiovasculardisease, stroke and heart failure; 4. Disorders or conditions in whichinflammatory reactions are involved, including atherosclerosis, chronicinflammatory bowel diseases (e.g., Crohn's disease and ulcerativecolitis), asthma, lupus erythematosus, arthritis, or other inflammatoryrheumatic disorders; 5. Disorders of cell cycle or cell differentiationprocesses such as adipose cell tumors, lipomatous carcinomas including,for example, liposarcomas, solid tumors, and neoplasms; 6.Neurodegenerative diseases and/or demyelinating disorders of the centraland peripheral nervous systems and/or neurological diseases involvingneuroinflammatory processes and/or other peripheral neuropathies,including Alzheimer's disease, multiple sclerosis, Parkinson's disease,progressive multifocal leukoencephalopathy and Guillian-Barre syndrome;7. Skin and dermatological disorders and/or disorders of wound healingprocesses, including erythemato-squamous dermatoses; and 8. Otherdisorders such as syndrome X, osteoarthritis, and acute respiratorydistress syndrome.

As used herein, the term “hyperglycemic” or “hyperglycemia,” when usedin reference to a condition of a subject means a transient or chronicabnormally high level of glucose present in the blood of a subject. Thecondition can be caused by a delay in glucose metabolism or absorptionsuch that the subject exhibits glucose intolerance or a state ofelevated glucose not typically found in normal subjects (e.g., inglucose-intolerant pre-diabetic subjects at risk of developing diabetes,or in diabetic subjects). Fasting plasma glucose (FPG) levels fornormoglycemia are less than about 100 mg/dl, for impaired glucosemetabolism, between about 100 and 126 mg/dl, and for diabetics greaterthan about 126 mg/dl.

As disclosed herein, the invention includes methods of preventing (e.g.,in subjects predisposed to having a particular disorder(s)), delaying,slowing or inhibiting progression of, the onset of, or treating (e.g.,ameliorating) obesity or an undesirable body mass (e.g., a greater thannormal body mass index, or “BMI” relative to an appropriate matchedsubject of comparable age, gender, race, etc.). Thus, in variousembodiments, a method of the invention for, for example, treatingobesity or an undesirable body mass (including the co-morbid conditionsof obesity, e.g., obstructive sleep apnea, arthritis, cancer (e.g.,breast, endometrial, and colon), gallstones or hyperglycemia, includescontacting or administering a peptide of the invention as set forthherein (e.g., a variant or fusion of FGF19 and/or FGF21 as set forth inTables 1-8 or SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70,71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70,M71, M72 and M73, respectively), for example) in an amount effective totreat obesity or an undesirable body mass. In particular aspects, asubject has a body mass index greater than 25, for example, 25-30,30-35, 35-40, or greater than 40.

Moreover, the invention includes methods of preventing (e.g., insubjects predisposed to having a particular disorder(s)), slowing orinhibiting the progression of, delaying the onset of, or treatingundesirable levels or abnormally elevated serum/plasma LDL, VLDL,triglycerides or cholesterol, all of which, alone or in combination, canlead to, for example, plaque formation, narrowing or blockage of bloodvessels, and increased risk of hypertension, stroke and coronary arterydisease. Such disorders can be due to, for example, geneticpredisposition or diet, for example.

The term “subject” refers to an animal. Typically, the animal is amammal that would benefit from treatment with a peptide sequence of theinvention. Particular examples include primates (e.g., humans), dogs,cats, horses, cows, pigs, and sheep.

Subjects include those having a disorder, e.g., a hyperglycemicdisorder, such as diabetes, or subjects that do not have a disorder butmay be at risk of developing the disorder, e.g., pre-diabetic subjectshaving FPG levels greater than 100 mg/dl, for example, between about 100and 126 mg/dl. Subjects at risk of developing a disorder include, forexample, those whose diet may contribute to development of acute orchronic hyperglycemia (e.g., diabetes), undesirable body mass orobesity, as well as those which may have a family history or geneticpredisposition towards development of acute or chronic hyperglycemia, orundesirable body mass or obesity.

As disclosed herein, treatment methods include contacting oradministering a peptide of the invention as set forth herein (e.g., avariant or fusion of FGF19 and or FGF21 as set forth in Tables 1-8 orSEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73(M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 andM73, respectively), for example) in an amount effective to achieve adesired outcome or result in a subject. A treatment that results in adesired outcome or result includes decreasing, reducing or preventingseverity or frequency of one or more symptoms of the condition in thesubject, e.g., an improvement in the subject's condition or a“beneficial effect” or “therapeutic effect.” Therefore, treatment candecrease or reduce or prevent the severity or frequency of one or moresymptoms of the disorder, stabilize or inhibit progression or worseningof the disorder, and in some instances, reverse the disorder,transiently (e.g., for 1-6, 6-12, or 12-24 hours), for medium term(e.g., 1-6, 6-12, 12-24 or 24-48 days) or long term (e.g., for 1-6,6-12, 12-24, 24-48 weeks, or greater than 24-48 weeks). Thus, in thecase of a hyperglycemic disorder, for example, treatment can lower orreduce blood glucose, improve glucose tolerance, improve glucosemetabolism, provide normal glucose homeostasis, lower or reduce insulinresistance, lower or reduce insulin levels, or decrease, prevent,improve, or reverse metabolic syndrome, or a histopathological changeassociated with or that results from the hyperglycemic disorder, such asdiabetes.

For example, a peptide sequence, method or use can lower or reduceglucose in one or more subjects having FPG levels greater than 100mg/dl, for example, between about 100 and 125 mg/dl, or greater than 125mg/dl, by 5-10%, 10-20%, 20-30%, or 30-50%, or more, or for example fromgreater than 200 mg/dl to less than 200 mg/dl, for greater than 150mg/dl to less than 150 mg/dl, from greater than 125 mg/dl to less than125 mg/dl, etc. In addition, a peptide sequence, method or use can loweror reduce glucose, for example, for pre-diabetes or for diabetes (e.g.,Type 2) subjects with baseline HbAIc levels greater than about 5%, 6%,7%, 8%, 9% or 10%, in particular 5%, 6%, or 7%.

Non-limiting examples of an improvement of a histopathological changeassociated with a hyperglycemic condition include, for example,decreasing, inhibiting, reducing or arresting: the destruction ordegeneration of pancreas cells (e.g., β-cells), kidney damage such astubule calcification or nephropathy, degeneration of liver, eye damage(e.g., diabetic retinopathy, cataracts), diabetic foot, ulcerations inmucosa such as mouth and gums, periodontitis, excess bleeding, slow ordelayed healing of injuries or wounds (e.g., that lead to diabeticcarbuncles), skin infections and other cutaneous disorders,cardiovascular and coronary heart disease, peripheral vascular disease,stroke, dyslipidemia, hypertension, obesity, or the risk of developingany of the foregoing. Improvement in undesirable body mass or obesitycan include, for example, a reduction of body mass (as reflected by BMIor the like) or an improvement in an associated disorder, such as adecrease in triglyceride, cholesterol, LDL or VLDL levels, a decrease inblood pressure, a decrease in intimal thickening of the blood vessel, adecreased or reduced risk of cardiovascular disease, or stroke, decreasein resting heart rate, etc.

An “effective amount” or a “sufficient amount” for use and/or fortreating a subject refer to an amount that provides, in single ormultiple doses, alone, or in combination with one or more othercompositions (therapeutic agents such as a drug or treatment forhyperglycemia), treatments, protocols, or therapeutic regimens agents, adetectable response of any duration of time (transient, medium or longterm), a desired outcome in or an objective or subjective benefit to asubject of any measurable or detectable degree or for any duration oftime (e.g., for hours, days, months, years, or cured). Such amountstypically are effective to ameliorate a disorder, or one, multiple orall adverse symptoms, consequences or complications of the disorder, toa measurable extent, although reducing or inhibiting a progression orworsening of the disorder, is considered a satisfactory outcome.

As used herein, the term “ameliorate” means an improvement in thesubject's disorder, a reduction in the severity of the disorder, or aninhibition of progression or worsening of the disorder (e.g.,stabilizing the disorder). In the case of a hyperglycemic disorder(e.g., diabetes, insulin resistance, glucose intolerance, metabolicsyndrome, etc.), for example, an improvement can be a lowering or areduction in blood glucose, a reduction in insulin resistance, areduction in glucagon, an improvement in glucose tolerance, or glucosemetabolism or homeostasis. An improvement in a hyperglycemic disorderalso can include improved pancreatic function (e.g., inhibit or preventβ-cell/islet destruction or enhance β-cell number and/or function), adecrease in a pathology associated with or resulting from the disorder,such as an improvement in histopathology of an affected tissue or organ,as set forth herein. In the case of undesirable body mass or obesity,for example, an improvement can be a decrease in weight gain, areduction of body mass (as reflected in reduced BMI, for example) or animprovement in a condition associated with undesirable body massobesity, for example, as set forth herein (e.g., a lowering or areduction of blood glucose, triglyceride, cholesterol, LDL or VLDLlevels, a decrease in blood pressure, a decrease in intimal thickeningof the blood vessel, etc.).

A therapeutic benefit or improvement therefore need not be completeablation of any one, most or all symptoms, complications, consequencesor underlying causes associated with the disorder or disease. Thus, asatisfactory endpoint is achieved when there is a transient, medium orlong term, incremental improvement in a subject's condition, or apartial reduction in the occurrence, frequency, severity, progression,or duration, or inhibition or reversal, of one or more associatedadverse symptoms or complications or consequences or underlying causes,worsening or progression (e.g., stabilizing one or more symptoms orcomplications of the condition, disorder or disease), of the disorder ordisease, over a duration of time (hours, days, weeks, months, etc.).

Thus, in the case of a disorder treatable by a peptide sequence of theinvention, the amount of peptide sufficient to ameliorate a disorderwill depend on the type, severity and extent, or duration of thedisorder, the therapeutic effect or outcome desired, and can be readilyascertained by the skilled artisan. Appropriate amounts will also dependupon the individual subject (e.g., the bioavailability within thesubject, gender, age, etc.). For example, a transient, or partial,restoration of normal glucose homeostasis in a subject can reduce thedosage amount or frequency of insulin injection, even though completefreedom from insulin has not resulted.

An effective amount can be ascertained, for example, by measuring one ormore relevant physiological effects. In a particular non-limitingexample in the case of a hyperglycemic condition, a lowering orreduction of blood glucose or an improvement in glucose tolerance testcan be used to determine whether the amount of invention peptidesequence, including subsequences, sequence variants and modified formsof the exemplified peptide sequences (e.g., sequences listed in Tables1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72and M73, respectively)) is effective to treat a hyperglycemic condition.In another particular non-limiting example, an effective amount is anamount sufficient to reduce or decrease any level (e.g., a baselinelevel) of FPG, wherein, for example, an amount sufficient to reduce aFPG level greater than 200 mg/dl to less than 200 mg/dl, an amountsufficient to reduce a FPG level between 175 mg/dl and 200 mg/dl to lessthan the pre-administration level, an amount sufficient to reduce a FPGlevel between 150 mg/dl and 175 mg/dl to less than thepre-administration level, an amount sufficient to reduce a FPG levelbetween 125 mg/dl and 150 mg/dl to less than the pre-administrationlevel, and so on (e.g., reducing FPG levels to less than 125 mg/dl, toless than 120 mg/dl, to less than 115 mg/dl, to less than 110 mg/dl,etc.). In the case of HbAIc levels, an effective amount includes anamount sufficient to reduce or decrease levels by more than about 10% to9%, by more than about 9% to 8%, by more than about 8% to 7%, by morethan about 7% to 6%, by more than about 6% to 5%, and so on. Moreparticularly, a reduction or decrease of HbAIc levels by about 0.1%,0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%,10%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, or more is an effective amountin accordance with the invention. In yet another particular non-limitingexample in the case of undesirable body mass or obesity, an effectiveamount is an amount sufficient to decrease or reduce the body mass index(BMI) of a subject, a decrease or reduction of glucose, a decrease orreduction in serum/plasma levels of triglyceride, lipid, cholesterol,fatty acids, LDL and/or VLDL. In yet further particular non-limitingexamples, an amount is an amount sufficient to decrease or reduce any ofthe aforementioned parameters by, for example, about 0.1%, 0.25%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%,33%, 35%, 40%, 45%, 50%, or more.

Methods and uses of the invention for treating a subject are applicablefor prophylaxis to prevent a disorder in a subject, such as ahyperglycemic disorder, or development of undesirable body mass orobesity. Alternatively, methods and uses can be practiced during orfollowing treatment of a subject. For example, prior to, during orfollowing treatment of a subject to lower glucose using insulin oranother glucose lowering drug or therapeutic agent, for example, amethod or use of the invention can, for example, a peptide sequence ofthe invention can be administered to the subject. In addition, acomposition such as a peptide sequence of the invention can be combinedwith another drug or agent, such as a glucose lowering drug ortherapeutic agent, for example.

Accordingly, methods and uses of the invention for treating a subjectcan be practiced prior to, substantially contemporaneously with orfollowing another treatment, and can be supplemented with other forms oftherapy. Supplementary therapies include other glucose loweringtreatments, such as insulin, an insulin sensitivity enhancer and otherdrug treatments, a change in diet (low sugar, fats, etc.), weight losssurgery—(reducing stomach volume by gastric bypass, gastrectomy),gastric banding, gastric balloon, gastric sleeve, etc. For example, amethod or use of the invention for treating a hyperglycemic or insulinresistance disorder can be used in combination with drugs or otherpharmaceutical compositions that lower glucose or increase insulinsensitivity in a subject. Drugs for treating diabetes include, forexample, biguanides and sulphonylureas (e.g., tolbutamide,chlorpropamide, acetohexamide, tolazamide, glibenclamide and glipizide),thiazolidinediones (rosiglitazone, pioglitazone), GLP-1 analogues,Dipeptidyl peptidase-4 (DPP-4) inhibitors, bromocriptine formulations(e.g. and bile acid sequestrants (e.g., colesevelam), and insulin (bolusand basal analogs), metformin (e.g., metformin hydrochloride) with orwithout a thiazolidinedione (TZD), and SGLT-2 inhibitors. Appetitesuppression drugs are also well known and can be used in combinationwith the methods of the invention. Supplementary therapies can beadministered prior to, contemporaneously with or following inventionmethods and uses.

Peptide sequences of the invention including subsequences, sequencevariants and modified forms of the exemplified peptide sequences(sequences listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50,51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51,M52, M53, M69, M70, M71, M72 and M73, respectively)), may be formulatedin a unit dose or unit dosage form. In a particular embodiment, apeptide sequence is in an amount effective to treat a subject in need oftreatment, e.g., due to hyperglycemia. Exemplary unit doses range fromabout 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000,25,000-50,000 ng; from about 25-250, 250-500, 500-1000, 1000-2500 or2500-5000, 5000-25,000, 25,000-50,000 μg; and from about 25-250,250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000, 25,000-50,000mg.

Peptide sequences of the invention including subsequences, sequencevariants and modified forms of the exemplified peptide sequences(sequences listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50,51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51,M52, M53, M69, M70, M71, M72 and M73, respectively)) can be administeredto provide the intended effect as a single dose or multiple dosages, forexample, in an effective or sufficient amount. Exemplary doses rangefrom about 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000,5000-25,000, 25,000-50,000 pg/kg; from about 50-500, 500-5000,5000-25,000 or 25,000-50,000 ng/kg; and from about 25-250, 250-500,500-1000, 1000-2500 or 2500-5000, 5000-25,000, 25,000-50,000 μg/kg.Single or multiple doses can be administered, for example, multipletimes per day, on consecutive days, alternating days, weekly orintermittently (e.g., twice per week, once every 1, 2, 3, 4, 5, 6, 7 or8 weeks, or once every 2, 3, 4, 5 or 6 months).

Peptide sequences of the invention including subsequences, variants andmodified forms of the exemplified peptide sequences (sequences listed inTables 1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70,71, 72 and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70,M71, M72 and M73, respectively)) can be administered and methods may bepracticed via systemic, regional or local administration, by any route.For example, a peptide sequence can be administered parenterally (e.g.,subcutaneously, intravenously, intramuscularly, or intraperitoneally),orally (e.g., ingestion, buccal, or sublingual), inhalation,intradermally, intracavity, intracranially, transdermally (topical),transmucosally or rectally. Peptide sequences of the invention includingsubsequences, variants and modified forms of the exemplified peptidesequences (sequences listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5,48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49,M50, M51, M52, M53, M69, M70, M71, M72 and M73, respectively)) andmethods of the invention including pharmaceutical compositions can beadministered via a (micro)encapsulated delivery system or packaged intoan implant for administration.

The invention further provides “pharmaceutical compositions,” whichinclude a peptide sequence (or sequences) of the invention, includingsubsequences, variants and modified forms of the exemplified peptidesequences (sequences listed in Tables 1-8 and SEQ ID NOs: 1, 2, 3, 5,48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2, M3, M5, M48, M49,M50, M51, M52, M53, M69, M70, M71, M72 and M73, respectively)), and oneor more pharmaceutically acceptable or physiologically acceptablediluent, carrier or excipient. In particular embodiments, a peptidesequence or sequences are present in a therapeutically acceptableamount. The pharmaceutical compositions may be used in accordance withthe invention methods and uses. Thus, for example, the pharmaceuticalcompositions can be administered ex vivo or in vivo to a subject inorder to practice treatment methods and uses of the invention.

Pharmaceutical compositions of the invention can be formulated to becompatible with the intended method or route of administration;exemplary routes of administration are set forth herein. In addition,the pharmaceutical compositions may further comprise othertherapeutically active agents or compounds disclosed herein (e.g.,glucose lowering agents) or known to the skilled artisan which can beused in the treatment or prevention of various diseases and disorders asset forth herein.

Pharmaceutical compositions typically comprise a therapeuticallyeffective amount of at least one of the peptide sequences of theinvention, including subsequences, variants and modified forms of theexemplified peptide sequences (sequences listed in Tables 1-8 and SEQ IDNOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72 and 73 (M1, M2,M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72 and M73,respectively)) and one or more pharmaceutically and physiologicallyacceptable formulation agents. Suitable pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients include, butare not limited to, antioxidants (e.g., ascorbic acid and sodiumbisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethylor n-propyl, p-hydroxybenzoate), emulsifying agents, suspending agents,dispersing agents, solvents, fillers, bulking agents, buffers, vehicles,diluents, and/or adjuvants. For example, a suitable vehicle may bephysiological saline solution or citrate buffered saline, possiblysupplemented with other materials common in pharmaceutical compositionsfor parenteral administration. Neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. Those skilled in theart will readily recognize a variety of buffers that could be used inthe pharmaceutical compositions and dosage forms used in the invention.Typical buffers include, but are not limited to pharmaceuticallyacceptable weak acids, weak bases, or mixtures thereof. Buffercomponents also include water soluble materials such as phosphoric acid,tartaric acids, lactic acid, succinic acid, citric acid, acetic acid,ascorbic acid, aspartic acid, glutamic acid, and salts thereof.

A primary solvent in a vehicle may be either aqueous or non-aqueous innature. In addition, the vehicle may contain other pharmaceuticallyacceptable excipients for modifying or maintaining the pH, osmolarity,viscosity, sterility or stability of the pharmaceutical composition. Incertain embodiments, the pharmaceutically acceptable vehicle is anaqueous buffer. In other embodiments, a vehicle comprises, for example,sodium chloride and/or sodium citrate.

Pharmaceutical compositions of the invention may contain still otherpharmaceutically-acceptable formulation agents for modifying ormaintaining the rate of release of an invention peptide. Suchformulation agents include those substances known to artisans skilled inpreparing sustained release formulations. For further referencepertaining to pharmaceutically and physiologically acceptableformulation agents, see, for example, Remington's PharmaceuticalSciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages1435-1712, The Merck Index, 12th Ed. (1996, Merck Publishing Group,Whitehouse, N.J.); and Pharmaceutical Principles of Solid Dosage Forms(1993, Technonic Publishing Co., Inc., Lancaster, Pa.). Additionalpharmaceutical compositions appropriate for administration are known inthe art and are applicable in the methods and compositions of theinvention.

A pharmaceutical composition may be stored in a sterile vial as asolution, suspension, gel, emulsion, solid, or dehydrated or lyophilizedpowder. Such compositions may be stored either in a ready to use form, alyophilized form requiring reconstitution prior to use, a liquid formrequiring dilution prior to use, or other acceptable form. In someembodiments, a pharmaceutical composition is provided in a single-usecontainer (e.g., a single-use vial, ampoule, syringe, or autoinjector(similar to, e.g., an EpiPen®)), whereas a multi-use container (e.g., amulti-use vial) is provided in other embodiments. Any drug deliveryapparatus may be used to deliver invention peptides, including implants(e.g., implantable pumps) and catheter systems, both of which are knownto the skilled artisan. Depot injections, which are generallyadministered subcutaneously or intramuscularly, may also be utilized torelease invention peptides over a defined period of time. Depotinjections are usually either solid- or oil-based and generally compriseat least one of the formulation components set forth herein. The skilledartisan is familiar with possible formulations and uses of depotinjections.

A pharmaceutical composition can be formulated to be compatible with itsintended route of administration. Thus, pharmaceutical compositionsinclude carriers, diluents, or excipients suitable for administration byroutes including parenteral (e.g., subcutaneous (s.c.), intravenous,intramuscular, or intraperitoneal), intradermal, oral (e.g., ingestion),inhalation, intracavity, intracranial, and transdermal (topical).

Pharmaceutical compositions may be in the form of a sterile injectableaqueous or oleagenous suspension. This suspension may be formulatedusing suitable dispersing or wetting agents and suspending agentsdisclosed herein or known to the skilled artisan. The sterile injectablepreparation may also be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent, for example, as asolution in 1,3-butane diol. Acceptable diluents, solvents anddispersion media that may be employed include water, Ringer's solution,isotonic sodium chloride solution, Cremophor EL™ (BASF, Parsippany,N.J.) or phosphate buffered saline (PBS), ethanol, polyol (e.g.,glycerol, propylene glycol, and liquid polyethylene glycol), andsuitable mixtures thereof. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. Moreover, fatty acids such as oleic acid find use in thepreparation of injectables. Prolonged absorption of particularinjectable formulations can be achieved by including an agent thatdelays absorption (e.g., aluminum monostearate or gelatin).

Pharmaceutical compositions may be in a form suitable for oral use, forexample, as tablets, capsules, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules, or syrups, solutions, microbeads or elixirs. Pharmaceuticalcompositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions. Such compositions may contain one or more agents such assweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets containing an invention peptide may be inadmixture with non-toxic pharmaceutically acceptable excipients suitablefor the manufacture of tablets. These excipients include, for example,diluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc.

Tablets, capsules and the like suitable for oral administration may beuncoated or they may be coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed. They may also be coated by techniques known in the art toform osmotic therapeutic tablets for controlled release. Additionalagents include biodegradable or biocompatible particles or a polymericsubstance such as polyesters, polyamine acids, hydrogel, polyvinylpyrrolidone, polyanhydrides, polyglycolic acid, ethylene-vinylacetate,methylcellulose, carboxymethylcellulose, protamine sulfate, orlactide/glycolide copolymers, polylactide/glycolide copolymers, orethylenevinylacetate copolymers in order to control delivery of anadministered composition. For example, the oral agent can be entrappedin microcapsules prepared by coacervation techniques or by interfacialpolymerization, by the use of hydroxymethylcellulose orgelatin-microcapsules or poly (methylmethacrolate) microcapsules,respectively, or in a colloid drug delivery system. Colloidal dispersionsystems include macromolecule complexes, nano-capsules, microspheres,microbeads, and lipid-based systems, including oil-in-water emulsions,micelles, mixed micelles, and liposomes. Methods for preparation of suchformulations are known to those skilled in the art and are commerciallyavailable.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture thereof. Such excipients aresuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents may be a naturally-occurring phosphatide, for examplelecithin, or condensation products of an alkylene oxide with fattyacids, for example polyoxy-ethylene stearate, or condensation productsof ethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified herein.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example, liquidparaffin, or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example, gum acacia or gum tragacanth;naturally-occurring phosphatides, for example, soy bean, lecithin, andesters or partial esters derived from fatty acids; hexitol anhydrides,for example, sorbitan monooleate; and condensation products of partialesters with ethylene oxide, for example, polyoxyethylene sorbitanmonooleate.

Pharmaceutical compositions can also include carriers to protect thecomposition against rapid degradation or elimination from the body, suchas a controlled release formulation, including implants, liposomes,hydrogels, prodrugs and microencapsulated delivery systems. For example,a time delay material such as glyceryl monostearate or glyceryl stearatealone, or in combination with a wax, may be employed. Prolongedabsorption of injectable pharmaceutical compositions can be achieved byincluding an agent that delays absorption, for example, aluminummonostearate or gelatin. Prevention of the action of microorganisms canbe achieved by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and thelike.

The invention also includes invention peptides in the form ofsuppositories for rectal administration. The suppositories can beprepared by mixing an invention peptide with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials include, but are not limited to, cocoa butter andpolyethylene glycols.

In accordance with the invention, there are provided methods ofidentifying a peptide (or a subsequence, variant or modified form as setforth herein) having glucose lowering activity without substantialhepatocellular carcinoma (HCC) activity. In one embodiment, a methodincludes: screening (e.g., assaying or measuring) a peptide sequence (ora subsequence, variant or modified form as set forth herein) for glucoselowering activity; and screening (e.g., assaying or measuring) a peptidesequence (or a subsequence, variant or modified form as set forthherein) for HCC activity, or expression of a marker correlating with HCCactivity. A peptide having glucose lowering activity and reduced orabsent HCC activity thereby identifies the peptide. In particularaspects, the marker correlating with HCC activity comprises lipidprofile—a peptide that has less lipid increasing activity compared toFGF19 indicates the peptide has reduced or absent HCC activity; or themarker correlating with HCC activity comprises aldo-keto reductase geneexpression—a peptide that down-regulates or decreases aldo-ketoreductase gene expression compared to FGF19 indicates that the peptidehas reduced or absent HCC activity; or the marker indicative of HCCactivity comprises Slc1a2 gene expression—a peptide that up-regulates orincreases Slc1a2 gene expression compared to FGF19 indicates that thepeptide has reduced or absent HCC activity.

The terms “assaying” and “measuring” and grammatical variations thereofare used interchangeably herein and refer to either qualitative orquantitative determinations, or both qualitative and quantitativedeterminations. When the terms are used in reference to detection, anymeans of assessing the relative amount is contemplated, including thevarious methods set forth herein and known in the art. For example, geneexpression can be assayed or measured by a Northern blot, Western blot,immunoprecipitation assay, or by measuring activity, function or amountof the expressed protein (e.g., aldo-keto reductase or Slc1a2).

Risk factors for HCC, the most common type of liver cancer, include type2 diabetes (probably exacerbated by obesity). The risk of HCC in type 2diabetics is greater (from ˜2.5 to ˜7 times the non-diabetic risk)depending on the duration of diabetes and treatment protocol.

Various methodologies can be used in the screening and diagnosis of HCCand are well known to the skilled artisan. Indicators for HCC includedetection of a tumor maker such as elevated alpha-fetoprotein (AFP) ordes-gamma carboxyprothrombin (DCP) levels. A number of differentscanning and imaging techniques are also helpful, including ultrasound,CT scans and MRI. In relation to the invention, evaluation of whether apeptide (e.g., a candidate peptide) exhibits evidence of inducing HCCmay be determined in vivo by, for example, quantifying HCC noduleformation in an animal model, such as db/db mice, administered apeptide, compared to HCC nodule formation by wild type FGF19.Macroscopically, liver cancer may be nodular, where the tumor nodules(which are round-to-oval, grey or green, well circumscribed but notencapsulated) appear as either one large mass or multiple smallermasses. Alternatively, HCC may be present as an infiltrative tumor whichis diffuse and poorly circumscribed and frequently infiltrates theportal veins.

Pathological assessment of hepatic tissue samples is generally performedafter the results of one or more of the aforementioned techniquesindicate the likely presence of HCC. Thus, methods of the invention mayfurther include assessing a hepatic tissue sample from an in vivo animalmodel (e.g., a db/db mouse) useful in HCC studies in order to determinewhether a peptide sequence exhibits evidence of inducing HCC. Bymicroscopic assessment, a pathologist can determine whether one of thefour general architectural and cytological types (patterns) of HCC arepresent (i.e., fibrolamellar, pseudoglandular (adenoid), pleomorphic(giant cell) and clear cell).

The invention also includes the generation and use of antibodies, andfragments thereof, that bind the peptide sequences of the invention,including subsequences, sequence variants and modified forms of theexemplified peptide sequences (including the peptides listed in Tables1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72and M73, respectively)).

As used herein, the terms “antibodies” (Abs) and “immunoglobulins” (Igs)refer to glycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to an antigen, immunoglobulinsinclude both antibodies and other antibody-like molecules which may lackantigen specificity.

The term “antibody” includes intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies)formed from at least two intact antibodies, and antibody bindingfragments including Fab and F(ab)′₂, provided that they exhibit thedesired biological activity. The basic antibody structural unitcomprises a tetramer, and each tetramer is composed of two identicalpairs of polypeptide chains, each pair having one “light” chain (about25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminalportion of each chain includes a variable region of about 100 to 110 ormore amino acids primarily responsible for antigen recognition. Incontrast, the carboxy-terminal portion of each chain defines a constantregion primarily responsible for effector function. Human light chainsare classified as kappa and lambda light chains, whereas human heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Bindingfragments are produced by recombinant DNA techniques, or by enzymatic orchemical cleavage of intact antibodies. Binding fragments include Fab,Fab′, F(ab′)₂, Fv, and single-chain antibodies.

Each heavy chain has at one end a variable domain (VH) followed by anumber of constant domains. Each light chain has a variable domain atone end (VL) and a constant domain at its other end; the constant domainof the light chain is aligned with the first constant domain of theheavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. The antibody chains all exhibit the samegeneral structure of relatively conserved framework regions (FR) joinedby three hyper-variable regions, also called complementarity-determiningregions or CDRs. The CDRs from the two chains of each pair are alignedby the framework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

An intact antibody has two binding sites and, except in bifunctional orbispecific antibodies, the two binding sites are the same. A bispecificor bifunctional antibody is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a population of substantially homogeneous antibodies, thatis, the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. In contrast to polyclonal antibodypreparations which include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen.

A “neutralizing antibody” is an antibody molecule that is able toeliminate or significantly reduce an effector function of a targetantigen to which it binds.

Antibody binding fragments may be produced by enzymatic or chemicalcleavage of intact antibodies. Digestion of antibodies with the enzymepapain results in two identical antigen-binding fragments, also known as“Fab” fragments, and an “Fc” fragment which has no antigen-bindingactivity. Digestion of antibodies with the enzyme pepsin results in aF(ab′)₂ fragment in which the two arms of the antibody molecule remainlinked and comprise two-antigen binding sites. The F(ab′)₂ fragment hasthe ability to crosslink antigen.

The term “Fab” refers to a fragment of an antibody that comprises theconstant domain of the light chain and the CH1 domain of the heavychain. The term “Fv” when used herein refers to the minimum fragment ofan antibody that retains both antigen-recognition and antigen-bindingsites. In a two-chain Fv species, this region consists of a dimer of oneheavy-chain and one light-chain variable domain in non-covalentassociation. In a single-chain Fv species, one heavy-chain and onelight-chain variable domain can be covalently linked by a flexiblepeptide linker such that the light and heavy chains can associate in a“dimeric” structure analogous to that in a two-chain Fv species. It isin this configuration that the three CDRs of each variable domaininteract to define an antigen-binding site on the surface of the VH-VLdimer. While the six CDRs, collectively, confer antigen-bindingspecificity to the antibody, even a single variable domain (or half ofan Fv comprising only three CDRs specific for an antigen) has theability to recognize and bind antigen.

The term “complementarity determining regions” or “CDRs” refers to partsof immunological receptors that make contact with a specific ligand anddetermine its specificity. The term “hypervariable region” refers to theamino acid residues of an antibody which are responsible forantigen-binding. The hypervariable region generally comprises amino acidresidues from a “complementarity determining region” or “CDR” and/orthose residues from a “hypervariable loop”.

As used herein, the term “epitope” refers to binding sites forantibodies on protein antigens. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains, as well as specific three dimensional structural andcharge characteristics. An antibody is said to bind an antigen when thedissociation constant is ≦1 μM, preferably ≦100 nM, and most preferably≦10 nM. An increased equilibrium constant (“K_(D)”) means that there isless affinity between the epitope and the antibody, whereas a decreasedequilibrium constant means that there is a higher affinity between theepitope and the antibody. An antibody with a K_(D) of “no more than” acertain amount means that the antibody will bind to the epitope with thegiven K_(D) or more strongly. Whereas K_(D) describes the bindingcharacteristics of an epitope and an antibody, “potency” describes theeffectiveness of the antibody itself for a function of the antibody.There is not necessarily a correlation between an equilibrium constantand potency; thus, for example, a relatively low K_(D) does notautomatically mean a high potency.

The term “selectively binds” in reference to an antibody does not meanthat the antibody only binds to a single substance, but rather that theK_(D) of the antibody to a first substance is less than the K_(D) of theantibody to a second substance. An antibody that exclusively binds to anepitope only binds to that single epitope.

When administered to humans, antibodies that contain rodent (murine orrat) variable and/or constant regions are sometimes associated with, forexample, rapid clearance from the body or the generation of an immuneresponse by the body against the antibody. In order to avoid theutilization of rodent-derived antibodies, fully human antibodies can begenerated through the introduction of human antibody function into arodent so that the rodent produces fully human antibodies. Unlessspecifically identified herein, “human” and “fully human” antibodies canbe used interchangeably herein. The term “fully human” can be usefulwhen distinguishing antibodies that are only partially human from thosethat are completely, or fully human. The skilled artisan is aware ofvarious methods of generating fully human antibodies.

In order to address possible human anti-mouse antibody responses,chimeric or otherwise humanized antibodies can be utilized. Chimericantibodies have a human constant region and a murine variable region,and, as such, human anti-chimeric antibody responses may be observed insome patients. Therefore, it is advantageous to provide fully humanantibodies against multimeric enzymes in order to avoid possible humananti-mouse antibody or human anti-chimeric antibody responses.

Fully human monoclonal antibodies can be prepared, for example, by thegeneration of hybridoma cell lines by techniques known to the skilledartisan. Other preparation methods involve the use of sequences encodingparticular antibodies for transformation of a suitable mammalian hostcell, such as a CHO cell. Transformation can be by any known method forintroducing polynucleotides into a host cell, including, for example,packaging the polynucleotide in a virus (or into a viral vector) andtransducing a host cell with the virus (or vector) or by transfectionprocedures known in the art. Methods for introducing heterologouspolynucleotides into mammalian cells are well known in the art andinclude dextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei. Mammalian cell lines available ashosts for expression are well known in the art and include, but are notlimited to CHO cells, HeLa cells, and human hepatocellular carcinomacells.

Antibodies can be used diagnostically and/or therapeutically. Forexample, the antibodies can be used as a diagnostic by detecting thelevel of one or more peptides of the invention in a subject, and eithercomparing the detected level to standard control level or to a baselinelevel in a subject determined previously (e.g., prior to any illness).The antibodies can be used as a therapeutic to modulate the activity ofone or more peptides of the invention, thereby having an effect on acondition or disorder.

The invention provides kits including, but not limited to, peptidesequences of the invention, optionally in combination with one or moretherapeutic agents, compositions and pharmaceutical compositionsthereof, packaged into suitable packaging material. A kit optionallyincludes a label or packaging insert including a description of thecomponents or instructions for use in vitro, in vivo, or ex vivo, of thecomponents therein. Exemplary instructions include instructions forreducing or lowering blood glucose, treatment of hyperglycemia,treatment of diabetes, etc.

A kit can contain a collection of such components, e.g., two or morepeptide sequences alone, or a combination of a peptide sequence withanother therapeutically useful composition (e.g., an anti-diabetic drug,such as a gastrin compound).

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,vials, tubes, etc.).

Kits of the invention can include labels or inserts. Labels or insertsinclude “printed matter,” e.g., paper or cardboard, separate or affixedto a component, a kit or packing material (e.g., a box), or attached to,for example, an ampule, tube or vial containing a kit component. Labelsor inserts can additionally include a computer readable medium, such asa disk (e.g., hard disk, card, memory disk), optical disk such as CD- orDVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage mediasuch as RAM and ROM or hybrids of these such as magnetic/optical storagemedia, FLASH media or memory type cards.

Labels or inserts can include identifying information of one or morecomponents therein, dose amounts, clinical pharmacology of the activeingredient(s) including mechanism of action, pharmacokinetics andpharmacodynamics. Labels or inserts can include information identifyingmanufacturer information, lot numbers, manufacturer location and date.

Labels or inserts can include information on a condition, disorder,disease or symptom for which a kit component may be used. Labels orinserts can include instructions for the clinician or for a subject forusing one or more of the kit components in a method, treatment protocolor therapeutic regimen. Instructions can include dosage amounts,frequency or duration, and instructions for practicing any of themethods, treatment protocols or therapeutic regimes set forth herein.Exemplary instructions include instructions for treatment or use of apeptide sequence as set forth herein. Kits of the invention thereforecan additionally include labels or instructions for practicing any ofthe methods and uses of the invention described herein includingtreatment methods and uses.

Labels or inserts can include information on any benefit that acomponent may provide, such as a prophylactic or therapeutic benefit.Labels or inserts can include information on potential adverse sideeffects, such as warnings to the subject or clinician regardingsituations where it would not be appropriate to use a particularcomposition. Adverse side effects could also occur when the subject has,will be or is currently taking one or more other medications that may beincompatible with the composition, or the subject has, will be or iscurrently undergoing another treatment protocol or therapeutic regimenwhich would be incompatible with the composition and, therefore,instructions could include information regarding such incompatibilities.

Invention kits can additionally include other components. Each componentof the kit can be enclosed within an individual container and all of thevarious containers can be within a single package. Invention kits can bedesigned for cold storage. Invention kits can further be designed tocontain peptide sequences of the invention, or that contain nucleicacids encoding peptide sequences. The cells in the kit can be maintainedunder appropriate storage conditions until ready to use.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed herein.

All applications, publications, patents and other references, GenBankcitations and ATCC citations cited herein are incorporated by referencein their entirety. In case of conflict, the specification, includingdefinitions, will control. As used herein, the singular forms “a”,“and,” and “the” include plural referents unless the context clearlyindicates otherwise. Thus, for example, reference to “a peptidesequence” or a “treatment,” includes a plurality of such sequences,treatments, and so forth.

As used herein, numerical values are often presented in a range formatthroughout this document. The use of a range format is merely forconvenience and brevity and should not be construed as an inflexiblelimitation on the scope of the invention unless the context clearlyindicates otherwise. Accordingly, the use of a range expressly includesall possible subranges, all individual numerical values within thatrange, and all numerical values or numerical ranges including integerswithin such ranges and fractions of the values or the integers withinranges unless the context clearly indicates otherwise. This constructionapplies regardless of the breadth of the range and in all contextsthroughout this patent document. Thus, for example, reference to a rangeof 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%,91-95%, 91-94%, 91-93%, and so forth. Reference to a range of 90-100%also includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%,91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%,etc., and so forth.

In addition, reference to a range of 1-3, 3-5, 5-10, 10-20, 20-30,30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120,120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200,200-225, 225-250 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, etc. In a further example, reference to a rangeof 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000,5000-50,000 includes any numerical value or range within or encompassingsuch values, e.g., 25, 26, 27, 28, 29 . . . 250, 251, 252, 253, 254 . .. 500, 501, 502, 503, 504 . . . , etc.

As also used herein a series of ranges are disclosed throughout thisdocument. The use of a series of ranges include combinations of theupper and lower ranges to provide another range. This constructionapplies regardless of the breadth of the range and in all contextsthroughout this patent document. Thus, for example, reference to aseries of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75,75-100, 100-150, includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75,5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40,20-50, 20-75, 20-100, 20-150, and so forth.

For the sake of conciseness, certain abbreviations are used herein. Oneexample is the single letter abbreviation to represent amino acidresidues. The amino acids and their corresponding three letter andsingle letter abbreviations are as follows:

alanine Ala (A) arginine Arg (R) asparagine Asn (N) aspartic acid Asp(D) cysteine Cys (C) glutamic acid Glu (E) glutamine Gln (Q) glycine Gly(G) histidine His (H) isoleucine Ile (I) leucine Leu (L) lysine Lys (K)methionine Met (M) phenylalanine Phe (F) proline Pro (P) serine Ser (S)threonine Thr (T) tryptophan Trp (W) tyrosine Tyr (Y) valine Val (V)

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis. Thus, even thoughthe invention is generally not expressed herein in terms of what theinvention does not include, aspects that are not expressly included inthe invention are nevertheless disclosed herein.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLES Example 1

The following is a description of various methods and materials used inthe studies herein.

Animals.

db/db mice were purchased from The Jackson Laboratory (Bar Habor, Me.),Mice were kept in accordance with welfare guidelines under controlledlight (12 hr light and 12 hr dark cycle, dark 6:30 pm-6:30 am),temperature (22±4° C.) and humidity (50%±20%) conditions. They had freeaccess to water (autoclaved distilled water) and were fed ad libitum ona commercial diet (Harlan Laboratories, Indianapolis, Ind., Irradiated2018 Teklad Global 18% Protein Rodent Diet) containing 17 kcal % fat, 23kcal % protein and 60 kcal % carbohydrate. For diet-induced obesity,C57BL6/J mice (Jackson Laboratory) were maintained on a high-fat diet(D12492, Research Diet, New Brunswick, N.J. USA) containing 60 kcal %fat, 20 kcal % protein and 20 kcal % carbohydrate for 16-20 weeks. Allanimal studies were approved by the NGM Institutional Animal Care andUse Committee.

DNA and Amino Acid Sequences.

cDNA of ORF encoding human FGF19 (Homo sapiens FGF19, GenBank AccessionNo. NM_005117.2) variants

Protein sequence encoded by the cDNA (GenBank Accession No. NP_005108.1)PCR. FGF19 ORF was amplified with polymerase chain reaction (PCR) usingrecombinant DNA (cDNA) prepared from human small intestinal tissue. PCRreagents kits with Phusion high-fidelity DNA polymerase were purchasedfrom New England BioLabs (F-530L, Ipswich, Mass.). The following primerswere used: forward PCR primer: 5′ CCGACTAGTCACCatgcggagcgggtgtgtgg andreverse PCR primer: 5′ ATAAGAATGCGGCCGCTTACTTCTCAAAGCTGGGACTCCTC.

Amplified DNA fragment was digested with restriction enzymes Spe I andNot I (the restriction sites were included in the 5′ or 3′ PCR primers,respectively) and was then ligated with AAV transgene vectors that hadbeen digested with the same restriction enzymes. The vector used forexpression contained a selectable marker and an expression cassettecomposed of a strong eukaryotic promoter 5′ of a site for insertion ofthe cloned coding sequence, followed by a 3′ untranslated region andbovine growth hormone polyadenylation tail. The expression construct isalso flanked by internal terminal repeats at the 5′ and 3′ ends.

Production and Purification of AAV.

AAV293 cells (obtained from Agilent Technologies, Santa Clara, Calif.)were cultured in Dulbeco's Modification of Eagle's Medium (DMEM,Mediatech, Inc. Manassas, Va.) supplemented with 10% fetal bovine serumand 1× antibiotic-antimycotic solution (Mediatech, Inc. Manassas, Va.).The cells were plated at 50% density on day 1 in 150 mm cell cultureplates and transfected on day 2, using calcium phosphate precipitationmethod with the following 3 plasmids (20 μg/plate of each): AAVtransgene plasmid, pHelper plasmids (Agilent Technologies) and AAV2/9plasmid (Gao et al., J. Virol. 78:6381 (2004)). 48 hours aftertransfection, the cells were scraped off the plates, pelleted bycentrifugation at 3000×g and resuspended in buffer containing 20 mM TrispH 8.5, 100 mM NaCl and 1 mM MgCl₂. The suspension was frozen in analcohol dry ice bath and was then thawed in 37° C. water bath. Thefreeze and thaw cycles were repeated three times; Benzonase®(Sigma-aldrich, St. Louis, Mo.) was added to 50 units/ml; deoxycholatewas added to a final concentration of 0.25%. After an incubation at 37°C. for 30 min, cell debris was pelleted by centrifugation at 5000×g for20 min. Viral particles in the supernatant were purified using adiscontinued iodixanal (Sigma-aldrich, St. Louis, Mo.) gradient aspreviously described (Zolotukhin S. et al (1999) Gene Ther. 6:973). Theviral stock was concentrated using Vivaspin® 20 (MW cutoff 100,000Dalton, Sartorius Stedim Biotech, Aubagne, France) and re-suspended inphosphate-buffered saline (PBS) with 10% glycerol and stored at −80° C.To determine the viral genome copy number, 2 μl of viral stock wereincubated in 6 μl of solution containing 50 units/ml Benzonase®, 50 mMTris-HCl pH 7.5, 10 mM MgCl₂ and 10 mM CaCl₂ at 37° C. for 30 minutes.

Afterwards, 15 μl of the solution containing 2 mg/ml of Proteinase K,0.5% SDS and 25 mM EDTA were added and the mixture was incubated foradditional 20 min at 55° C. to release viral DNA. Viral DNA was cleanedwith mini DNeasy® Kit (Qiagen, Valencia, Calif.) and eluted with 40 μlof water. Viral genome copy (GC) was determined by using quantitativePCR.

Viral stock was diluted with PBS to desirable GC/ml. Viral workingsolution (200 μl) was delivered into mice via tail vein injection.

Blood Glucose Assay.

Blood glucose in mouse tail snip was measured using ACCU-CHEK Activetest strips read by ACCU-CHEK Active meter (Roche Diagnostics,Indianapolis, Ind.) following manufacturer's instruction.

Lipid Profile Assay.

Whole blood from mouse tail snips was collected into plain capillarytubes (BD Clay Adams SurePrep™, Becton Dickenson and Co. Sparks, Md.).Serum and blood cells were separated by spinning the tubes in anAutocrit™ Ultra 3 (Becton Dickinson and Co. Sparks, Md.). Serum sampleswere assayed for lipid profile (triglyceride, total cholesterol, HDL,and non-HDL) using Integra™ 400 Clinical Analyzer (Roche Diagnostics,Indianapolis, Ind.) following the manufacturer's instructions.

Serum FGF19/FGF21/Variants Exposure Level Assay.

Whole blood (about 50 μl/mouse) from mouse tail snips was collected intoplain capillary tubes (BD Clay Adams SurePrep, Becton Dickenson and Co.Sparks, Md.). Serum and blood cells were separated by spinning the tubesin an Autocrit™ Ultra 3 (Becton Dickinson and Co. Sparks, Md.). FGF19,FGF21, and variant exposure levels in serum were determined using EIAkits (Biovendor) by following the manufacturer's instructions.

Hepatocellular Carcinoma (HCC) Assay.

Liver specimen was harvested from db/db mice 6 months after AAVinjection. HCC score is recorded as the number of HCC nodules on thesurface of the entire liver from variants-injected mice divided by thenumber of HCC nodules from wildtype FGF19-injected mice.

Liver Gene Expression Assay.

Liver specimen was harvested and homogenized in TRIzol® reagent(Invitrogen). Total RNA was extracted following manufacturer'sinstruction. RNA was treated with DNase (Ambion) followed byquantitative RT-PCR analysis using TaqMan® primers and reagents fromApplied Biosystems. Relative mRNA levels of aldo-keto reductase andslc1a2 in the liver was calculated using ΔΔCt method.

FGFR4 Binding and Activity Assays.

Solid phase ELISA (binding) and ERK phosphorylation assay were performedusing purified recombinant proteins. FGFR binding assay was conductedusing solid phase ELISA. Briefly, 96 well plate was coated with 2 ug/mlanti-hFc antibody and incubated with 1 ug/ml FGFR1-hFc or FGFR4-hFc.Binding to FGF19 variants in the presence of 1 ug/ml soluble b-klothoand 20 ug/ml heparin were detected by biotinylated anti-FGF19 antibodies(0.2 ug/mL), followed by streptavidin-HRP incubation (100 ng/mL). ForFGFR4 activation assay, Hep3B cells were stimulated with FGF19 variantsfor 10 minutes at 37 C, then immediately lysed and assayed for ERKphosphorylation using a commercially available kit from Cis-Bio.

Example 2

The following is a description of studies showing the glucose loweringactivity of various sequence variants of FGF19 and FGF21, andFGF19/FGF21 fusion constructs.

FIG. 1 illustrates exemplary FGF19/FGF21 fusion constructs, and thesegments from each of FGF19 and FGF21 present in the fusion peptides.These peptides were analyzed for glucose lowering activity andstatistically significant lipid elevating or increasing activity (Tables1-8 and SEQ ID NOs: 1, 2, 3, 5, 48, 49, 50, 51, 52, 53, 69, 70, 71, 72and 73 (M1, M2, M3, M5, M48, M49, M50, M51, M52, M53, M69, M70, M71, M72and M73, respectively)).

Mice (db/db) were injected with viral vector expressing FGF19, FGF21 orvariants, and analyzed after injection. Glucose-lowering activity ofeach sequence is represented by a “+” symbol (a “−” symbol means noglucose lowering activity, a “+/−” symbol means variants retain minimalglucose-lowering activity); lipid elevating activity is represented by a“+” symbol (a “−” symbol means no lipid elevating activity, a “+/−”symbol means variants retain minimal lipid-elevating activity, FIG. 1).

Two fusions of FGF21 and FGF19, denoted variant M5 and variant 45 (M45),exhibited glucose lowering activity and an absence of statisticallysignificant lipid elevating or increasing activity. Variants denoted M1,M2 and M69, respectively (SEQ ID NOs:1, 2 and 69, respectively), alsoexhibited glucose lowering activity (FIGS. 2B and 2C, Table 5). Datacomparing M5, M1, M2 and M69 glucose lowering activity and lipidelevating or increasing activity to FGF19 and FGF21 are illustrated inFIGS. 2A-2C and 3A-3C.

Example 3

The following is a description of studies showing that variants M5, M1,M2 and M69 are not tumorigenic, as determined by hepatocellularcarcinoma (HCC) formation, and that variants M5, M2 and M69 also do notreduce lean muscle and fat mass.

Animals (db/db) were injected with AAV vectors expressing FGF19, FGF21,M5, M1, M2, or M69, or injected with saline, and analyzed 6 months afterinjection. The data indicate that variants M5, M1, M2, and M69 did notinduce (HCC) formation significantly (FIGS. 4A-4C).

Animals (db/db mice) were also injected with viral vector expressingFGF19, FGF21, M5, M1, M2 or M69, or injected with saline, and analyzed 6months after injection for the effect of on lean mass and fat mass. Thedata indicate that M5, M2 and M69 peptides did not cause a statisticallysignificant reduction in lean mass or fat mass, in contrast to FGF21,and that M1 peptide reduces lean mass (FIGS. 5A-5C).

Example 4

The following is a data summary of 25 additional variant peptidesanalyzed for lipid elevating activity and tumorigenesis. The dataclearly show a positive correlation between lipid elevation andtumorigenesis, as determined by hepatocellular carcinoma (HCC) formationin db/db mice.

Tables 1 to 3 summarize data for 26 different variant peptides. Suchexemplified variant peptides have FGF19 C-terminal sequence:PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO:188) at theC-terminal portion, e.g., following the “TSG” amino acid residues.Notably, variant peptides (7 total, including M5) that did not cause astatistically significant elevation of lipids did not inducehepatocellular carcinoma (HCC) formation. In contrast, all variantpeptides (17 total) that caused a statistically significant elevation oflipids also caused hepatocellular carcinoma (HCC) formation in mice.This data indicates that there is a strong positive correlation betweenlipid elevating activity and hepatocellular carcinoma (HCC) formation.Accordingly, lipid elevating activity can be used as an indicator and/orpredictor of hepatocellular carcinoma (HCC) formation in animals.

TABLE 1Elevated Triglyceride and Cholesterol in db/db Mice Appears to Positively CorrelateWith HCC Formation.

SEQ ID NO: Core SEQ ID NO: Lipid Eleva- tion HCC Forma- tion FGF19RPLAFS--------------------DAGPHVHYGWGDPI 99 (aa 1-20) RLRHLYTSG 185 + +FGF21 HPIPDSSPLLQ--FGGQV 100 (aa 1-16) RQRYLYTDD 186 − − M5R-HPIPDSSPLLQ--FGGQV 5 (aa 1-17) RLRHLYTSG 185 − − M74R----------------DAGPHVHYGWGDPI 74 (aa 1-15) RLRHLYTSG 185 + + M75R---------------------------VHYGWGDPI 75 (aa 1-10) RLRHLYTSG 185 − − M76R------------------------------------GDPI 76 (11 1-5) RLRHLYTSG 185 − −M77 R-------------------------------------------- 77 (aa 1) RLRHLYTSG185 − − M78 R------------------AGPHVHYGWGDPI 78 (aa 1-14) RLRHLYTSG185 + + M79 R---------------------GPHVHYGWGDPI 79 (aa 1-13) RLRHLYTSG185 + + M80 R-----------------------PHVHYGWGDPI 80 (aa 1-12) RLRHLYTSG185 − − M81 R------------------------HVHYGWGDPI 81 (aa 1-11) RLRHLYTSG185 − −

TABLE 2Elevated Triglyceride and Cholesterol in db/db Mice Appears to Positively Correlatewith HCC Formation

SEQ ID NO: Core SEQ ID NO: Lipid Elevation HCC Formation FGF19RPLAFSDAGPHVHYGWGDPI 99 (aa 1-20) RLRHLYTSG 185 + + FGF21    HPIPDSSPLLQ--FGGQV 100 (1-16) RQRYLYTDD 186 − − M82RPLAFSAAGPHVHYGWGDPI 82 (aa 1-20) RLRHLYTSG 185 + + M83RPLAFSDAAPHVHYGWGDPI 83 (aa 1-20) RLRHLYTSG 185 +/− +/ M84RPLAFSDAGAHVHYGWGDPI 84 (aa 1-20) RLRHLYTSG 185 +/− +/ M85RPLAFSDAGPHVHYGAGDPI 85 (aa 1-20) RLRHLYTSG 185 − − M86RPLAFSDAGPHVHYGWGAPI 86 (aa 1-20) RLRHLYTSG 185 + + M87RPLAFSDAGPHVHYGWGDAI 87 (aa 1-20) RLRHLYTSG 185 + +

TABLE 3Elevated Triglyceride and Cholesterol in db/db Mice Appears to Positively Correlatewith HCC Formation

Core SEQ ID NO Lipid Elevation HCC Formation FGF19 RPLAFSDAGPHVHYGWGDPIRLRHLYTSG 99 (aa 1-29) + + FGF21   HPIPDSSPLLQ--FGGQV RQRYLYTDD100 (aa 1-25) − − H31A/S141A(M89) FGF19 + + H31A/H142A(M89) FGF19 + +K127A/R129A(M90) FGF19 + + K127A/S141A(M91) FGF19 + + K127A/H142A(M92)FGF19 + + R129A/S141A(M93) FGF19 + + S141A/H142A(M94) FGF19 + +K127A/H142A(M95) FGF19 + + K127A/R129A/S141A(M96) FGF19 + +K127A/R129A/H142A(M97) FGF19 + + K127A/R129A/S141A/H142A(M98) FGF19 + +

Example 5

The following is a data summary of additional FGF19 variant peptidesanalyzed for glucose lowering activity and lipid elevating activity.

Table 4 illustrates the peptide “core sequences” of 35 additional FGF19variants, denoted M5 to M40. Such exemplified variant peptides haveFGF19 C-terminal sequence,PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO:188) at theC-terminal portion, e.g., following the “TSG” amino acid residues of thecore sequence. The data clearly show that variants M6, M7, M8, mM38 andM39 have the desired characteristics of glucose lowering activity andnot statistically significant lipid elevating activity in db/db mice.

TABLE 4  Additional Variants and Fine Mapping of the N-terminal DomainSEQ ID Glucose Lipid N-terminal Domain SEQ ID NO: Core NO: LoweringElevation FGF19 RPLAFSDAGPHVHYGWGDPI  99 (aa 1-20) RLRHLYTSG 185 + +FGF21 HPIPDSSPLLQ--FGGQV 100 (aa 1-16) RQRYLYTDD 186 + − M5R-HPIPDSSPLLQ--FGGQV   5 (aa 1-17) RLRHLYTSG 185 + − M6R-------DSSPLLQ--FGGQV   6 (aa 1-18) RLRHLYTSG 185 + − M7RPLAFSDSSPLLQ--FGGQV   7 (aa 1-18) RLRHLYTSG 185 + − M8R-HPIPDSSPLLQ--WGDPI   8 (aa 1-17) RLRHLYTSG 185 + − M9R-HPIPDSSPLLQFGWGDPI   9 (aa 1-19) RLRHLYTSG 185 + + M10R-HPIPDSSPHVHYGWGDPI  10 (aa 1-19) RLRHLYTSG 185 − + M11RPLAFSDAGPLLQ--WGDPI  11 (aa 1-18) RLRHLYTSG 185 N/D N/D M12RPLAFSDAGPLLQFGWGDPI  12 (aa 1-20) RLRHLYTSG 185 − + M13RPLAFSDAGPLLQ--FGGQV  13 (aa 1-18) RLRHLYTSG 185 − − M14R-HPIPDSSPHVHYG--GQV  14 (aa 1-17) RLRHLYTSG 185 − − M15RPLAFSDAGPHVHYG--GQV  15 (aa 1-18) RLRHLYTSG 185 + + M16RPLAFSDAGPHVH--WGDPI  16 (aa 1-18) RLRHLYTSG 185 N/D N/D M17RPLAFSDAGPHV--GWGDPI  17 (aa 1-18) RLRHLYTSG 185 N/D N/D M18RPLAFSDAGPH--YGWGDPI  18 (aa 1-18) RLRHLYTSG 185 N/D N/D M19RPLAFSDAGP-V-YGWGDPI  19 (aa 1-18) RLRHLYTSG 185 N/D N/D M20RPLAFSDAGP-VH-GWGDPI  20 (aa 1-18) RLRHLYTSG 185 N/D N/D M21RPLAFSDAGP-VHY-WGDPI  21 (aa 1-18) RLRHLYTSG 185 N/D N/D M22RPLAFSDAGPHVH-GWGDPI  22 (aa 1-18) RLRHLYTSG 185 N/D N/D M23RPLAFSDAGPH-H-GWGDPI  23 (aa 1-18) RLRHLYTSG 185 N/D N/D M24RPLAFSDAGPH-HY-WGDPI  24 (aa 1-18) RLRHLYTSG 185 N/D N/D M25RPLAFSDAGPHV-Y-WGDPI  25 (aa 1-18) RLRHLYTSG 185 N/D N/D M26RPLAFSDSSPLVH--WGDPI  26 (aa 1-18) RLRHLYTSG 185 N/D N/D M27RPLAFSDSSPHVH--WGDPI  27 (aa 1-18) RLRHLYTSG 185 N/D N/D M28RPLAFSDAPHV---WGDPI  28 (aa 1-16) RLRHLYTSG 185 N/D N/D M29RPLAFSDAGPHVHY-WGDPI  29 (aa 1-19) RLRHLYTSG 185 N/D N/D M30RPLAFSDAGPHVHYAWGDPI  30 (aa 1-20) RLRHLYTSG 185 N/D N/D M31R-HPIPDSSPLLQ--FGAQV  31 (aa 1-17) RLRHLYTSG 185 +/− − M32R-HPIPDSSPLLQ--FGIYQV  32 (aa 1-18) RLRHLYTSG 185 − − M33R-HPIPDSSPLLQ--FGGQV  33 (aa 1-17) RLRHLYTSG 185 − − M34R-HPIPDSSPLLQ--FGGAV  34 (aa 1-17) RLRHLYTSG 185 +/− − M35R-HPIPDSSPLLQ--FGGEV  35 (aa 1-17) RLRHLYTSG 185 +/− +/  M36R-HPIPDSSPLLQ--FGGQV  36 (aa 1-17) RLRHLYTSG 185 +/− − M37R-HPIPDSSPLLQ--FGGQA  37 (aa 1-17) RLRHLYTSG 185 − − M38R-HPIPDSSPLLQ--FGGQI  38 (aa 1-17) RLRHLYTSG 185 + − M39R-HPIPDSSPLLQ--FGGQT  39 (aa 1-17) RLRHLYTSG 185 + − M40R-HPIPDSSPLLQFGWGQPV  40 (aa 1-19) RLRHLYTSG 185 − +

TABLE 4a

Core SEQ ID NO. Glucose Lowering Lipid Elevation HCC Formation FGF19RPLAFSDAGPHVHYGWGDPI RLRHLYTSG 99 (aa 1-29) + + + FGF21  HPIPDSSPLLQ--FGGQV RQRYLYTDD 100 (aa 1-25) + − − M5R-HPIPDSSPLLQ--FGGQV RLRHLYTSG 5 (aa 1-26) + − − M9 R-HPIPDSSPLLQFGWGDPIRLRHLYTSG 9 (aa 1-28) + + + M8 R-HPIPDSSPLLQ--WGDPI RLRHLYTSG8 (aa 1-26) + + + M12 RPLAFSDAGPLLQFGWGDPI RLRHLYTSG 12 (aa 1-29) − + +M10 R-HPIPDSSPHVHYGWGDPI RLRHLYTSG 10 (aa 1-28) − + + M13RPLAFSDAGPLLQ--FGGQV RLRHLYTSG 13 (aa 1-27) − + + M15RPLAFSDAGPHVHYG--GQV RLRHLYTSG 15 (aa 1-27) − − +/− M14R-HPIPDSSPHVHYG--GQV RLRHLYTSG 14 (aa 1-26) − − +/− M43RPLAFSDAGPHVHYG-GD-I RLRHLYTSG 43 (aa 1-27) + − +/− M6R-----DSSPLLQ--FGGQV RLRHLYTSG 6 (aa 1-22) + − − M7 RPLAFSDSSPLLQ--FGGQVRLRHLYTSG 7 (aa 1-27) − − −

TABLE 4b

Core SEQ ID NO. Glucose Lowering Lipid Elevation HCC Formation FGF19RPLAFSDAGPHVHYGWGDPI RLRHLYTSG 99 (aa 1-29) + + + FGF21  HPIPDSSPLLQ--FGGQV RQRYLTDD 100 (aa 1-25) + − − M5R-HPIPDSSPLLQ--FGGQV RLRHLYTSG 5 (aa 1-26) + − − M31R-HPIPDSSPLLQ--FGAQV RLRHLYTSG 31 (aa 1-26) + − + M32R-HPIPDSSPLLQ--FGDQV RLRHLYTSG 32 (aa 1-26) + − − M33R-HPIPDSSPLLQ--FGPQV RLRHLYTSG 33 (aa 1-26) − − + M34R-HPIPDSSPLLQ--FGGAV RLRHLYTSG 34 (aa 1-26) − − + M35R-HPIPDSSPLLQ--FGGEV RLRHLYTSG 35 (aa 1-26) − − + M36R-HPIPDSSPLLQ--FGGNV RLRHLYTSG 36 (aa 1-26) + − +/− M37R-HPIPDSSPLLQ--FGGQA RLRHLYTSG 37 (aa 1-26) − − + M38R-HPIPDSSPLLQ--FGGQI RLRHLYTSG 38 (aa 1-26) − − + M39R-HPIPDSSPLLQ--FGGQT RLRHLYTSG 39 (aa 1-26) − − + M40R-HPIPDSSPLLQFGWGQPV RLRHLYTSG 40 (aa 1-28) − + +

TABLE 4c

Core SEQ ID NO. Glucose Lowering Lipid Elevation HCC Formation FGF19RPLAFSDAGPHVHYGWGDPI RLRHLYTSG 99 (aa 1-29) + + + FGF21  HPIPDSSPLLQ--FGGQV RQRYLYTDD 100 (aa 1-25) + − − M5R-HPIPDSSPLLQ--FGGQV RLRHLYTSG 5 (aa 1-26) + − − M52R-----DSSPLLQ--WGDPI RLRHLYTSG 52 (aa 1-22) + + − M54RPLAFSDAGPLLQ--WGDPI RLRHLYTSG 54 (aa 1-27) − + + M55RPLAFSDAGPH--YGWGDPI RLRHLYTSG 55 (aa 1-27) − + + M56RPLAFSDAGP-V-YGWGDPI RLRHLYTSG 56 (aa 1-27) − + + M57RPLAFSDAGP-VT-GWGDPI RLRHLYTSG 57 (aa 1-27) − + + M58RPLAFSDAGP-VHY-WGDPI RLRHLYTSG 58 (aa 1-27) − + + M59RPLAFSDAGPH-H-GWGDPI RLRHLYTSG 59 (aa 1-27) − + + M60RPLAFSDAGPH-HY-WGDPI RLRHLYTSG 60 (aa 1-27) − + + M61RPLAFSDAGPHV--GWGDPI RLRHLYTSG 61 (aa 1-27) − + + M62RPLAFSDAGPHV-Y-WGDPI RLRHLYTSG 62 (aa 1-27) − + + M63RPLAFSDAGPHVH--WGDPI RLRHLYTSG 63 (aa 1-27) + + + M64RPLAFSDSSPLVH--WGDPI RLRHLYTSG 64 (aa 1-27) + + + M65RPLAFSDSSPHVH--WGDPI RLRHLYTSG 65 (aa 1-27) − + + M66RPLAFSDAGPHLQ--WGDPI RLRHLYTSG 66 (aa 1-27) + + + M67RPLAFSDAGPHV---WGDPI RLRHLYTSG 67 (aa 1-26) − − +/− M68RPLAFSDAGPHVHY-WGDPI RLRHLYTSG 68 (aa 1-28) − + − M4RPLAFSDAGPHVHYAWGDPI RLRHLYTSG 4 (aa 1-29) + + + M69R-----DSSPLVHYGWGDPI RLRHLYTSG 69 (aa 1-24) + + − M70MR----DSSPLVHYGWGDPI RLRHLYTSG 70 (aa 1-25) + + − M53M-----DSSPLLQ--WGDPI RLRHLYTSG 192 (aa 1-22) + + −

Table 5 illustrates the peptide sequences of 3 additional FGF19variants, denoted M1, M2 and M69. The data clearly show that these threevariants have the desired characteristics of glucose lowering activityin db/db mice (FIGS. 2B and 2C). These three variants appear to elevatelipids in db/db mice FIGS. 3B and 3C).

TABLE 5  Additional VariantsM1: RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK  (SEQ ID NO: 1)M2: RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK  (SEQ ID NO: 2)M69: RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK  (SEQ ID NO: 69)

Example 6

The following is a data summary showing that FGF19 reduces body weightin diet-induced obese mice and in ob/ob mice, and liver tumor formationactivity and body weight in db/db mice.

Mice were injected with FGF19 or FGF21 in AAV vector. Body weight wasrecorded 4 weeks after injection.

TABLE 6FGF19 reduces body weight in diet-induced obese mice and in ob/ob mice

core Body Weight- Lowering in DIO Body Weight- Lowering in ob/ob FGF19RPLAFSDAGPHVHYGWGDPI RLRHLYTSG 99 (aa 1-29) Increased FGF21  HPIPDSSPLLQ--FGGQV RQRYLYTDD 100 (aa 1-25) Decreased

TABLE 7Correlation of body weight and liver tumor formation of FGF19, FGF21 and selectedvariants in db/db mice

Core SEQ ID NO Liver Tumor Nodule Body Weight FGF19 RPLAFSDAGPHVHYGWGDPIRLRHLYTSG 99 (aa 1-29) + Increased FGF21   HPIPDSSPLLQ--FGGQV RQRYLYTDD100 (aa 1-25) − Decreased M5 R-HPIPDSSPLLQ--FGGQV RLRHLYTSG 5 (aa 1-26)− Increased M6 R-----DSSPLLQ--FGGQV RLRHLYTSG 6 (aa 1-22) − DecreasedM32 R-HPIPDSSPLLQ--FGDQV RLRHLYTSG 32 (aa 1-26) − Decreased M52R-----DSSPLLQ--WGDPI RLRHLYTSG 52 (aa 1-22) − Decreased M69R-----DSSPLVHYGWGDPI RLRHLYTSG 69 (aa 1-24) − Increased

Example 7

The following is a study showing that variant M5 and variant M69peptides reduce blood glucose.

Mice (ob/ob) were injected (subcutaneously) with M5 (0.1 and 1 mg/kg,s.c.) or FGF19 (1 mg/kg, s.c.), or variant M69 (0.1 and 1 mg/kg, s.c.)or FGF19 (1 mg/kg, s.c.). Plasma glucose levels were measured at 2, 4,7, and 24 hours after injection, and the results are shown in FIG. 6. M5(FIG. 6A) and variant M69 (FIG. 6B) showed similar glucose loweringeffects as wild type FGF19.

Example 8

This example describes a study showing that liver expression ofaldo-keto reductase family 1, member C18 (Akr1C18) and solute carrierfamily 1, member 2 (slc1a2) appears to correlate with HCC activity.

Mice (db/db) were injected with viral vector expressing FGF19 (HCC+),FGF21 (HCC−), dN2 (HCC−) or M5 (HCC−), or injected with GFP. Liversamples were harvested and analyzed by quantitative RT-PCR 2 weeks afterinjection. The data, shown in FIG. 7, shows that liver expression ofAkr1C18 and slc1a2 appears to correlate with HCC activity.

TABLE 8 Summary of FGF19 Variants in 3T3L1 Adipocyte Signaling AssayP-Erk assay in 3T3L1 adipocytes FGF19 FGF21 M5 M2 M63 M64 M1 M8Experiment#1: Emax 3.67 4.33 3.52 4.19 3.21 3.67 4.24 4.16 EC50 (nM)0.05 0.65 0.03 0.05 0.92 0.02 0.03 0.03 Experiment#2: Emax 4.52 4.834.01 5.56 4.17 4.85 5.30 5.34 EC50 (nM) 0.33 1.48 0.14 0.15 0.66 0.120.09 0.09 Experiment#3: Emax 4.09 4.14 3.74 4.24 3.15 4.15 4.77 4.16EC50 (nM) 0.16 1.50 0.24 0.14 0.28 0.14 0.07 0.14

Sequence Listing

The present specification is being filed with a computer readable form(CRF) copy of the Sequence Listing. The CRF entitled13370-048-999_SEQLIST.txt, which was created on Dec. 24, 2015 and is234,866 bytes in size, is incorporated herein by reference in itsentirety.

1.-74. (canceled)
 75. A method of reducing glucose levels in a subject,comprising administering to the subject an effective amount of a peptidehaving an amino acid sequence comprising or consisting of(SEQ ID NO: 69) RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK.


76. The method of claim 75, wherein the peptide has an amino acidsequence comprising SEQ ID NO:69.
 77. The method of claim 75, whereinthe peptide has an amino acid sequence consisting of SEQ ID NO:69. 78.The method of claim 76, wherein the peptide is fused with animmunoglobulin Fc region.
 79. The method of claim 77, wherein thepeptide is fused with an immunoglobulin Fc region.
 80. The method ofclaim 76, wherein the peptide is formulated as a pharmaceuticalcomposition, wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier.
 81. The method of claim 76, whereinthe method further comprises administration of a supplementary therapy.82. The method of claim 81, wherein said supplemental therapy is aweight loss surgery, gastric bypass, gastrectomy, gastric banding,gastric balloon, or gastric sleeve.
 83. The method of claim 81, whereinsaid supplemental therapy is a glucose lowering agent, insulin, GLP1analogue, biguanide, sulphonylurea, thiazolidinedione, a dipeptidylpeptidase-4 (DPP-4) inhibitor, a bromocriptine formulation, a bile acidsequestrant, metformin, a thiazolidinedione (TZD), a SGLT-2 inhibitor,or any combination thereof.
 84. The method of claim 83, wherein saidbiguanide or sulphonylurea is selected from the group consisting oftolbutamide, chlorpropamide, acetohexamide, tolazamide, glibenclamide,glipizide or any combination thereof.
 85. The method of claim 83,wherein said thiazolidinedione is rosiglitazone or pioglitazone, orcombination thereof.
 86. The method of claim 83, wherein said bile acidsequestrant is colesevelam.
 87. The method of claim 83, wherein saidinsulin is bolus insulin, basal insulin, or an analogue thereof.
 88. Themethod of claim 81, wherein said supplemental therapy is administeredprior to, contemporaneously with or following said method.
 89. Themethod of claim 76, wherein the glucose levels are blood glucose levels.90. The method of claim 76, wherein the subject has a metabolicdisorder.
 91. The method of claim 76, wherein the subject has an insulinresistance disorder.
 92. The method of claim 76, wherein the subject hashyperinsulinemia.
 93. The method of claim 76, wherein the subject hasglucose intolerance.
 94. The method of claim 76, wherein the subject hasa hyperglycemic disorder or is at risk of developing a hyperglycemicdisorder.
 95. The method of claim 76, wherein the subject has diabetes.96. The method of claim 76, wherein the subject has insulin-dependent(type I) diabetes.
 97. The method of claim 76, wherein the subject hastype II diabetes.
 98. The method of claim 76, wherein the subject hasgestational diabetes.
 99. The method of claim 76, wherein the subject isobese.
 100. The method of claim 76, wherein the subject has nonalcoholicfatty liver disease (NAFLD).
 101. The method of claim 76, wherein thesubject has nonalcoholic steatohepatitis (NASH).
 102. The method ofclaim 76, wherein the subject has a fasting plasma glucose (FPG) levelof greater than 100 mg/dl.
 103. The method of claim 76, wherein thesubject has an FPG level of 125 mg/dl or greater.
 104. The method ofclaim 76, wherein the subject has an FPG level between 100 and 125mg/dl.
 105. The method of claim 76, wherein the glucose levels arereduced by at least 5%.
 106. The method of claim 76, wherein the subjecthas a hemoglobin A1c (HbA1c) level above 6%.
 107. The method of claim77, wherein the peptide is formulated as a pharmaceutical composition,wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier.
 108. The method of claim 77,wherein the method further comprises administration of a supplementarytherapy.
 109. The method of claim 108, wherein said supplemental therapyis a weight loss surgery, gastric bypass, gastrectomy, gastric banding,gastric balloon, or gastric sleeve.
 110. The method of claim 108,wherein said supplemental therapy is a glucose lowering agent, insulin,GLP1 analogue, biguanide, sulphonylurea, thiazolidinedione, a DPP-4inhibitor, a bromocriptine formulation, a bile acid sequestrant,metformin, a TZD, a SGLT-2 inhibitor, or any combination thereof. 111.The method of claim 110, wherein said biguanide or sulphonylurea isselected from the group consisting of tolbutamide, chlorpropamide,acetohexamide, tolazamide, glibenclamide, glipizide or any combinationthereof.
 112. The method of claim 110, wherein said thiazolidinedione isrosiglitazone or pioglitazone, or combination thereof.
 113. The methodof claim 110, wherein said bile acid sequestrant is colesevelam. 114.The method of claim 110, wherein said insulin is bolus insulin, basalinsulin, or an analogue thereof.
 115. The method of claim 110, whereinsaid supplemental therapy is administered prior to, contemporaneouslywith or following said method.
 116. The method of claim 77, wherein theglucose levels are blood glucose levels.
 117. The method of claim 77,wherein the subject has a metabolic disorder.
 118. The method of claim77, wherein the subject has an insulin resistance disorder.
 119. Themethod of claim 77, wherein the subject has hyperinsulinemia.
 120. Themethod of claim 77, wherein the subject has glucose intolerance. 121.The method of claim 77, wherein the subject has a hyperglycemic disorderor is at risk of developing a hyperglycemic disorder.
 122. The method ofclaim 77, wherein the subject has diabetes.
 123. The method of claim 77,wherein the subject has insulin-dependent (type I) diabetes.
 124. Themethod of claim 77, wherein the subject has type II diabetes.
 125. Themethod of claim 77, wherein the subject has gestational diabetes. 126.The method of claim 77, wherein the subject is obese.
 127. The method ofclaim 77, wherein the subject has NAFLD.
 128. The method of claim 77,wherein the subject has NASH.
 129. The method of claim 77, wherein thesubject has a FPG level of greater than 100 mg/dl.
 130. The method ofclaim 77, wherein the subject has an FPG level of 125 mg/dl or greater.131. The method of claim 77, wherein the subject has an FPG levelbetween 100 and 125 mg/dl.
 132. The method of claim 77, wherein theglucose levels are reduced by at least 5%.
 133. The method of claim 77,wherein the subject has a HbA1c level above 6%.
 134. The method of claim78, wherein the peptide is formulated as a pharmaceutical composition,wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier.
 135. The method of claim 78,wherein the method further comprises administration of a supplementarytherapy.
 136. The method of claim 135, wherein said supplemental therapyis a weight loss surgery, gastric bypass, gastrectomy, gastric banding,gastric balloon, or gastric sleeve.
 137. The method of claim 135,wherein said supplemental therapy is a glucose lowering agent, insulin,GLP1 analogue, biguanide, sulphonylurea, thiazolidinedione, a DPP-4inhibitor, a bromocriptine formulation, a bile acid sequestrant,metformin, a TZD, a SGLT-2 inhibitor, or any combination thereof. 138.The method of claim 137, wherein said biguanide or sulphonylurea isselected from the group consisting of tolbutamide, chlorpropamide,acetohexamide, tolazamide, glibenclamide, glipizide or any combinationthereof.
 139. The method of claim 137, wherein said thiazolidinedione isrosiglitazone or pioglitazone, or combination thereof.
 140. The methodof claim 137, wherein said bile acid sequestrant is colesevelam. 141.The method of claim 137, wherein said insulin is bolus insulin, basalinsulin, or an analogue thereof.
 142. The method of claim 135, whereinsaid supplemental therapy is administered prior to, contemporaneouslywith or following said method.
 143. The method of claim 78, wherein theglucose levels are blood glucose levels.
 144. The method of claim 78,wherein the subject has a metabolic disorder.
 145. The method of claim78, wherein the subject has an insulin resistance disorder.
 146. Themethod of claim 78, wherein the subject has hyperinsulinemia.
 147. Themethod of claim 78, wherein the subject has glucose intolerance. 148.The method of claim 78, wherein the subject has a hyperglycemic disorderor is at risk of developing a hyperglycemic disorder.
 149. The method ofclaim 78, wherein the subject has diabetes.
 150. The method of claim 78,wherein the subject has insulin-dependent (type I) diabetes.
 151. Themethod of claim 78, wherein the subject has type II diabetes.
 152. Themethod of claim 78, wherein the subject has gestational diabetes. 153.The method of claim 78, wherein the subject is obese.
 154. The method ofclaim 78, wherein the subject has NAFLD.
 155. The method of claim 78,wherein the subject has NASH.
 156. The method of claim 78, wherein thesubject has a FPG level of greater than 100 mg/dl.
 157. The method ofclaim 78, wherein the subject has an FPG level of 125 mg/dl or greater.158. The method of claim 78, wherein the subject has an FPG levelbetween 100 and 125 mg/dl.
 159. The method of claim 78, wherein theglucose levels are reduced by at least 5%.
 160. The method of claim 78,wherein the subject has a HbA1c level above 6%.
 161. The method of claim79, wherein the peptide is formulated as a pharmaceutical composition,wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier.
 162. The method of claim 79,wherein the method further comprises administration of a supplementarytherapy.
 163. The method of claim 162, wherein said supplemental therapyis a weight loss surgery, gastric bypass, gastrectomy, gastric banding,gastric balloon, or gastric sleeve.
 164. The method of claim 162,wherein said supplemental therapy is a glucose lowering agent, insulin,GLP1 analogue, biguanide, sulphonylurea, thiazolidinedione, a DPP-4inhibitor, a bromocriptine formulation, a bile acid sequestrant,metformin, a TZD, a SGLT-2 inhibitor, or any combination thereof. 165.The method of claim 164, wherein said biguanide or sulphonylurea isselected from the group consisting of tolbutamide, chlorpropamide,acetohexamide, tolazamide, glibenclamide, glipizide or any combinationthereof.
 166. The method of claim 164, wherein said thiazolidinedione isrosiglitazone or pioglitazone, or combination thereof.
 167. The methodof claim 164, wherein said bile acid sequestrant is colesevelam. 168.The method of claim 164, wherein said insulin is bolus insulin, basalinsulin, or an analogue thereof.
 169. The method of claim 162, whereinsaid supplemental therapy is administered prior to, contemporaneouslywith or following said method.
 170. The method of claim 79, wherein theglucose levels are blood glucose levels.
 171. The method of claim 79,wherein the subject has a metabolic disorder.
 172. The method of claim79, wherein the subject has an insulin resistance disorder.
 173. Themethod of claim 79, wherein the subject has hyperinsulinemia.
 174. Themethod of claim 79, wherein the subject has glucose intolerance. 175.The method of claim 79, wherein the subject has a hyperglycemic disorderor is at risk of developing a hyperglycemic disorder.
 176. The method ofclaim 79, wherein the subject has diabetes.
 177. The method of claim 79,wherein the subject has insulin-dependent (type I) diabetes.
 178. Themethod of claim 79, wherein the subject has type II diabetes.
 179. Themethod of claim 79, wherein the subject has gestational diabetes. 180.The method of claim 79, wherein the subject is obese.
 181. The method ofclaim 79, wherein the subject has NAFLD.
 182. The method of claim 79,wherein the subject has NASH.
 183. The method of claim 79, wherein thesubject has a FPG level of greater than 100 mg/dl.
 184. The method ofclaim 79, wherein the subject has an FPG level of 125 mg/dl or greater.185. The method of claim 79, wherein the subject has an FPG levelbetween 100 and 125 mg/dl.
 186. The method of claim 79, wherein theglucose levels are reduced by at least 5%.
 187. The method of claim 79,wherein the subject has a HbA1c level above 6%.